Host transformed with yeast gene and ubiquitin/polypeptide fusions

ABSTRACT

A ubiquitin hydrolase is provided having a purity of at least 70% homogeneity based on the weight of the total protein in the composition. Also provided are DNA sequences encoding ubiquitin hydrolases, as well as expression systems for their recombinant production. Processes are provided for purification of a ubiquitin hydrolase from eukaryotes and for its use in recovering any desired polypeptide free from its fusion at its N-terminus with ubiquitin.

This is a divisional of copending application(s) Ser. No. 07/284,281filed on Dec. 14, 1988, now U.S. Pat. No. 5,108,919 which is acontinuation-in-part of co-pending application Ser. No. 07/210,909,filed on Jun. 24, 1988, now U.S. Pat. No. 5,156,968.

BACKGROUND OF THE INVENTION

This invention relates to the purification of ubiquitin hydrolaseshaving enzymatic activity in cleaving ubiquitin-protein conjugates. Thisinvention also relates to a process for preparing ubiquitin hydrolasesusing recombinant methods and a process for using same to isolatepolypeptides from fusions thereof with ubiquitin.

The polypeptide known as ubiquitin is highly conserved, has a molecularweight of 8,565, and contains 76 amino acid residues. It is encoded bygenes that contain varying numbers of the protein sequence repeatedwithout any stop codons between them or with other proteins. Ubiquitinis reviewed in Rechsteiner, Ann. Rev. Cell. Biol., 3: 1-30 (1987) and inRechsteiner, M., ed., Ubiquitin (New York: Plenum Press, 1988).

Ubiquitin was first purified during studies of peptides of the thymus.Radioimmunoassays for the peptide revealed that it was found widely inplant, animal, and yeast. Goldstein et al., Proc. Natl. Acad. Sci.U.S.A. 72: 11-15 (1975). The sequence of amino acids 1-74 of thymusubiquitin was determined by Schlesinger et al., Biochemistry, 14:2214-2218 (1975), revealing an NH₂ -terminal methionine and an arginineat position 74. The sequence was confirmed by Low et al., J. Biol.Chem., 254: 987-995 (1979). This form was later shown to be a degradedform of ubiquitin and is not active in its biological function. Theactive form is the 76 amino acid form. Wilkinsion and Audhya, J. Biol.Chem., 256: 9235-9241 (1981).

It has been found that ubiquitin is involved in the energy-dependentdegradation of intracellular proteins. Ganoth et al., J. Biol. Chem.,263: 12412-12419 (1988). Evidence exists that in eukaryotes, covalentconjugation of ubiquitin to the proteins is essential for theirselective degradation. Finley and Varshavsky, Trends Biochem. Sci., 10:343-346 (1985) and Finley et al., Cell, 37: 43 (1984).

Isopeptidases have been identified that are unique for eukaryotes. Theyare found to cleave in vitro an amide bond formed between a ubiquitinGly-COOH terminal and epsilon-NH₂ group of lysine on other polypeptides.For example, an isopeptidase was identified that cleaves the linkagebetween ubiquitin and lysozyme to yield free lysozyme. Hershko et al.,Proc. Natl. Acad. Sci. U.S.A., 81: 1619-1623 (1984). An isopeptidase wasalso detacted in reticulocyte extracts that cleaves ubiquitin-histone 2Aconjugates, with the release of undegraded histone. Andersen et al.,Biochemistry, 20: 1100-1104 (1981), Kanda et al., Biochim, Biophys.Acta, 870: 64-75 (1986), Matsui et al., J. Cell Biol., 95: (2) PA82(1982), and Matsui et al., Proc. Natl. Acad. Sci. U.S.A., 79: 1535-1539(1982). See also Hershko et al., Proc. Natl. Acad. Sci. U.S.A., 77:1783-1786 (1980) and Haas and Rose, Proc. Natl. Acad. Sci. U.S.A., 78:6845-6848 (1981).

Isopeptidase was purified 175-fold from calf thymus by ionexchangechromatography, gel filtration, and affinity chromatography on wholehistone and histone H2A Sepharose. Kanda et al., J. Cell Biol., 99: (4),PA135 (1984). This purified isopeptidase was found to be specific to theepsilon-(glycyl)lysine linkage in structural chromatin protein A24.

When the isopeptidase was purified, it was found to exist in growingChinese hamster cells as two major forms having molecular weight 250,000and 34,000, but was found to be present in human erythrocytes and calfthymus only in the 250,000 molecular weight form. These two forms ofenzyme were found to be distinct from one another, in that thedegradation of the large form did not result in the appearance of thesmaller form. Matsui, J. Cell Biol., 105: (4 Part 2), 187A (1987). Theauthor suggests that the large form is a stable constitutive enzyme andthat the small form with a rapid turnover rate is linked to themetabolic pathway of growth-related ubiquitin-protein conjugates.Isopeptidase activity of 30 kDa on silver-stained SDS-PAGE (calledcarboxyl-terminal hydrolase) was also identified in human red bloodcells. Pickart and Rose, J. Biol. Chem., 260: 7903-7910 (1985). The sameenzyme may be involved in the cleavage as is involved in processing theubiquitin-protein fusions. Pickart and Rose, J. Biol. Chem., 261:10210-10217 (1986). This enzyme was formerly called ubiquitincarboxyl-terminal esterase because it was found to hydrolyze ubiquitinesters of small thiols. Rose and Warms, Biochemistry, 22: 4234-4237(1983).

An activity of processing protease is reported in WO 88/02406 publishedApr. 7, 1988, in the context of designing or modifying protein structureat the protein or genetic level to produce specified amino termini basedon introducing the use of artificial ubiquitin-protein fusions.

Ubiquitin aldehyde was found to form strong complexes with mosthydrolases, e.g., the major ubiquitin-protein hydrolase of greater than200 KDa, a small 30 KDa cationic hydrolase, and the major hydrolase of30 KDa that acts on small molecule conjugates of ubiquitin. Rose et al.,Fed. Proc., 46: (6), 2087 (1987). It was concluded that ubiquitinhydrolases, in addition to being important in rescuing ubiquitin fromtraps with small nucleophiles, are necessary for recycling ubiquitinfrom protein conjugates that are only slowly degraded.

Recent analysis of the genes encoding ubiquitin from various organismsby molecular genetic techniques has shown that ubiquitin is synthesizedas a polyubiquitin precursor with multiple, contiguous stretches ofubiquitin sequences or as a protein fusion in which ubiquitin is locatedat the N-terminal domain of a larger protein. Ozkaynak et al., The EMBOJ., 6: 1429-1439 (1987); Lund et al., J. Biol. Chem., 260: 7609-7613(1985). The last copy of the ubiquitin sequence in the polyubiquitingene is usually followed by one amino acid extension after the uniqueArg-Gly-Gly terminus. Ozkaynak et al., supra.

Another discovery regarding cleavage of ubiquitin-protein conjugatesrevealed that when a chimeric gene encoding aubiquitin-beta-galactosidase fusion protein was expressed in yeast,ubiquitin was cleaved from the fusion protein, yielding adeubiquitinated beta-galactosidase. This endoproteolytic cleavage wasfound to take place regardless of the nature of the amino acid residueof beta-gel at the ubiquitin-beta-gal junction, with one exception.Bachmair et al., Science, 234: 179-186 (1986). It was also found thatdifferent residues could be exposed at the amino-termini of theotherwise identical beta-gal proteins. These authors suggested that thesame protease, as of then uncharacterized biochemically, was responsibleboth for the conversion of polyubiquitin into mature ubiquitin and forthe deubiquitination of the nascent ubiquitin-beta-gal protein.

Different investigators detected a proteolytic activity that convertedthe polyubiquitin to ubiquitin when a coupled in vitrotranscription/translation system was employed. Agell et al., J. CellBiol., 105: (4 pt 2), 82a (1987) and Agell et al., Proc. Natl. Acad.Sci., U.S.A., 85: 3693-3697 (1988). The polyubiquitin processingactivity was partially inhibited by ubiquitin aldehyde, a knowninhibitor of ubiquitin hydrolase. A purified preparation of thisproteolytic activity was found to be inactive, with further purificationof the putative protease then reported to be in progress.

At the American Chemical Society meeting on Sep. 26, 1988, chiron Corp.disclosed that fusion of a gene that has proven difficult to expressdirectly to a synthetic gene for yeast ubiquitin has allowed high-levelintracellular production of the desired protein as a mature polypeptide,cleaved in vivo by an endogenous yeast protease. See 1988 ACS AbstractBook, Abs. No. 34, P. J. Barr et al., "Production of Recombinant DNADerived Pharmaceuticals in the Yeast Saccharomyces cerevisiae."

Regarding the purification of substances using an irreversible step suchas cleavage, it was reported that fragments of proteins can be separatedby charge or size in one dimension and then a reagent used to alter theprotein fragments irreversibly for visualization in a second dimension.The objective of this work was to obtain amino acid sequence from theprotein. Hartley et al., Biochem. J., 80: 36 (1961).

In addition, it is known to recover and purify a protein from its fusionproduct with an "identification" peptide. EP 150,126 published Jul. 31,1985, equivalent to U.S. Pat. No. 4,703,004. In this process a hybridpolypeptide is synthesized with the identification peptide fused to adesired functional protein at the C-terminus of the identificationpeptide. The linking portion of the identification peptide is cleaved ata specific amino acid residue adjacent to the functional protein byusing a sequence-specific proteolytic enzyme or chemical agent. Thehybrid polypeptide is purified by affinity chromatography using animmobilized ligand specific to the antigenic portion of theidentification peptide. The protein is then cleaved from the isolatedhybrid polypeptide with an appropriate proteolytic agent to release themature functional protein.

Recovery of a product from its fusion using an identification peptidelinker or antibody is also disclosed. EP 35,384, published Sep. 9, 1981,and U.S. Pat. No. 4,732,852, issued Mar. 22, 1988. Moreover, recombinantproduction of polypeptides as fusion products with a charged amino acidpolymer, separating the fusion product from contaminants based on theproperties of the polymer, and cleaving the polymer from the fusionproduct using an exopeptidase has been reported. U.S. Pat. No.4,532,207, issued Jul. 30, 1985.

The major problem associated with cleaving fusion proteins produced byrecombinant means has been the lack of specific cleaving agents toremove the fusion protein moiety from the product protein in an exactand consistent manner. Chemical agents such as cyanogen bromide orhydroxylamine, or specific proteases such as Factor Xa or collagenase,that are used generally to achieve cleavage typically are onlycommercially practical in a limited number of protein fusion cleavages.

For example, if the specific amino acid that is required for thecleavage of a fusion protein (such as methionine for cyanogen bromide)is present internally in the amino acid sequence of the desired proteinproduct, the product will be clipped internally as well as cleaved fromthe fused polypeptide. For this reason and other reasons, the cleavingagents are generally specific only for one protein product. In addition,the cleavage itself may leave extra amino acid residues on the productprotein. Furthermore, almost all of the cleaving agents require extrarecovery steps to purify the more complex mixture that is generatedafter cleavage.

Accordingly, it is an object of the present invention to provide aubiquitin hydrolase that is purified to a sufficient degree that it canbe sequenced.

It is another object to provide quantities of a ubiquitin hydrolaseuseful for commercial purposes by using recombinant means to produce thehydrolase, free of source proteins.

It is still another object to provide a procedure for obtaining aheretofore unidentified yeast ubiquitin hydrolase.

It is another object to provide a method for producing and purifyingmature polypeptides, the method being characterized by removing thefusion protein moiety from the product moiety specifically andefficiently, by reducing the number and complexity of fusion recoverysteps, and by obtaining precise and reproducible cleavage of the productfree of extra unwanted terminal amino acid residues.

These and other objects will be obvious to those of ordinary skill inthe art.

SUMMARY OF THE INVENTION

In one aspect of the invention herein, these objects are achieved by acomposition comprising a ubiquitin hydrolase in a purity of at least 70%homogeneity based on the weight of the total protein in the composition.

In another aspect, the invention herein provides a process for purifyingubiquitin hydrolase comprising:

(a) homogenizing a eukaryotic cell fermentation paste and recovering theportion from the homogenate containing ubiquitin-hydrolase activity;

(b) salting out from the recovered hydrolase-containing portion of step(a) a precipitate containing ubiquitin-hydrolase activity;

(c) contacting a solution of the precipitate with an ion exchange resinand recovering the ubiquitin-hydrolase-active fraction;

(d) contacting a ubiquitin hydrolase-active fraction with a hydrophobicaffinity resin and recovering the ubiquitin-hydrolase-active fractionadsorbed to the resin;

(e) contacting a ubiquitin hydrolase-active fraction with an adsorptionchromatography resin and recovering the ubiquitin-hydrolase-activefraction adsorbed to the resin; and

(f) contacting a ubiquitin-hydrolase-active fraction with an ionexchange resin and recovering the hydrolase-active fraction.

In yet another aspect, the invention provides an isolated nucleic acidsequence comprising a sequence that encodes a ubiquitin hydrolase orfragments or variants thereof, an expression vector comprising thisnucleic acid sequence operably linked to control sequences recognized bya host transformed by the vector, and host cells transformed by such avector. The nucleic acid is preferably DNA, but can also be RNA or anRNA vector (retrovirus).

In a more specific aspect, the invention provides an isolated DNAsequence comprising a sequence that hybridizes under stringentconditions to the DNA sequence of FIG. 5 and that contains at leastabout ten nucleotides. Preferably the DNA sequence contains at leastabout twenty nucleotides, more preferably about thirty nucleotides, andmost preferably, about 40 nucleotides.

In yet another embodiment, the invention provides an isolated DNAsequence comprising a DNA sequence encoding an enzyme having an aminoacid sequence sufficiently duplicative of that of a ubiquitin hydrolaseto allow it to hydrolyze a ubiquitinpolypeptide conjugate at the amidebond linking the ubiquitin and polypeptide, thereby yielding intactpolypeptide with an unconjugated, mature N-terminus. The invention alsoprovides for expression vectors containing such DNA sequence operablylinked to appropriate control sequences, and hosts such as E. colitransformed with such vectors.

In a further aspect, the invention sets forth a method for in vitrocleavage of ubiquitin-polypeptide conjugates comprising:

(a) providing a ubiquitin-polypeptide conjugate in a compositioncomprising contaminant products of recombinant host cell culture,wherein the polypeptide is conjugated to the C-terminus of the ubiquitinand wherein the polypeptide contains any amino acid except proline atits N-terminus;

(b) contacting the composition with a reagent having specific affinityfor ubiquitin so that the conjugate is adsorbed on the reagent,separating the reagent and its absorbed conjugate from the rest of thecomposition, and recovering the conjugate from the reagent;

(c) contacting the recovered conjugate with ubiquitin hydrolase wherebythe conjugate is hydrolyzed to ubiquitin and mature polypeptide and theubiquitin hydrolase is immobilized; and

(d) contacting the material obtained from step (c) with a reagent havingspecific affinity for ubiquitin so that any residual conjugate and freeubiquitin are adsorbed on the reagent, and recovering the polypeptidefree from the reagent and the materials adsorbed thereon.

In still another aspect, the invention is directed to a method for invitro cleavage of ubiquitin-polypeptide conjugates comprising:

(a) providing a ubiquitin-polypeptide conjugate in a compositioncomprising contaminant products of recombinant host cell culture,wherein the polypeptide is conjugated to the C-terminus of the ubiquitinand wherein the polypeptide contains any amino acid except proline atits N-terminus;

(b) contacting the composition with a reagent having specific affinityfor ubiquitin so that the conjugate is adsorbed on the reagent andseparating the reagent and its adsorbed conjugate from the rest of thecomposition;

(d) contacting the reagent on which is adsorbed the conjugate withubiquitin hydrolase;

(e) separating the hydrolase and polypeptide from the reagent; and

(f) separating the polypeptide from the hydrolase.

In still another aspect, the invention provides a method for in vivocleavage of ubiquitin-polypeptide conjugates comprising:

(a) culturing prokaryotic host cells that have DNA encoding a ubiquitinhydrolase integrated into their chromosomes and are transformed with anexpression vector comprising a nucleotide sequence encoding aubiquitin-polypeptide conjugate wherein the polypeptide is conjugated tothe C-terminus of the ubiquitin and wherein the polypeptide contains anyamino acid except proline at its N-terminus such that the conjugate isexpressed; and

(b) recovering from the cultured cells the polypeptide free from theubiquitin to which it was conjugated.

In yet another aspect, the invention provides prokaryotic host cellshaving DNA encoding a ubiquitin hydrolase integrated into theirchromosomes. Most preferred of these are those host cells that are alsotransformed with an expression vector comprising a nucleotide sequenceencoding a ubiquitin-polypeptide conjugate wherein the polypeptide isconjugated to the C-terminus of the ubiquitin and wherein thepolypeptide contains any amino acid except proline at its N-terminus.

This invention also relates to an isolated yeast ubiquitin hydrolaseencoded by DNA that does not hybridize to the DNA sequence thathybridizes under stringent conditions to the DNA sequence of FIG. 5 andthat contains at least about ten nucleotides, preferably at least about20, more preferably at least about 30, and most preferably at leastabout 40 nucleotides. Similarly, the invention provides an isolatednucleic acid sequence encoding a ubiquitin hydrolase comprising anucleic acid sequence that does not hybridize to the above-identifiedDNA sequence. In addition, the invention furnishes an expression vectorcomprising this nucleic acid sequence operably linked to controlsequences recognized by a host transformed by the vector. Finally, theinvention provides a host cell transformed with this expression vector.

In further aspects, the invention provides an enzyme compositioncomprising the ubiquitin hydrolase of this invention in a buffer and akit comprising a ubiquitin hydrolase as one component and immobilizedanti-ubiquitin antibody as a second component.

The present invention makes it possible to produce a ubiquitin hydrolaseor derivatives thereof by recombinant techniques, as well as to provideproducts and methods related to such production. In addition, thisinvention enables a simplified and effective method for recoveringmature proteins and polypeptides from their fusion with another protein.Further, the invention allows variant polypeptides and proteins to beexpressed without concern about secretion or cleavage in undesiredpositions.

The in vivo cleavage technique herein permits the intracellularproduction of polypeptides from prokaryotes that are ordinarily unstablein the cell or that are not desired to be secreted, such as human growthhormone having no N-terminal methionine and γ-interferon. In the formercase, the N-terminal methionine need not be present during productionnor later removed after the protein is recovered from the medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (parts a-1, a-2, b-1 and b-2) depicts the sequence of a syntheticgene extending from XbaI to HindIII sites encoding a ubiquitin fusionpolypeptide wherein the polypeptide is the human (H2) relaxin B chain of32 amino acids (missing the N-terminal amino acid) linked at itsN-terminus to a hexapeptide. The hexapeptide is in turn linked at itsN-terminus to the C-terminus of ubiquitin. The six amino acids do notcode for any naturally occurring sequence.

FIG. 1b depicts the sequence of a synthetic gene extending from DraIIIto HindIII sites encoding a ubiquitin fusion polypeptide wherein thepolypeptide is the 33-amino-acid human (H2) relaxin B chain.

FIG. 1c depicts the construction of pT7-12TNF, an intermediate plasmidproviding the phi 10 promoter recognized by T7 polymerase (P_(T7)).

FIG. 1d depicts the construction from pT7-12TNF of several vectorscontaining ubiquitin fusion polypeptides used in the assay for theubiquitin hydrolase activity. The pTrpUbs-X series of plasmids arecommercially useful for producing ubiquitin fusion polypeptides in largequantities.

FIG. 2 is a schematic depiction of the ubiquitin hydrolase assay.

FIG. 3 (parts a-d) depicts the nucleotide and predicted amino acidsequence of a yeast ubiquitin hydrolase (designated herein as YUH-1).Predicted amino acids of the protein are shown below the DNA sequenceand are numbered from the first residue of the proposed N-terminus ofthe protein sequence. The figure also indicates the amino acid sequenceof the polypeptide used to derive Probe 1 for screening a yeast genomiclibrary to obtain a clone encoding a yeast ubiquitin hydrolase.

FIG. 4 depicts the orientation of the BamHI-SalI fragment in M13mp18 andM13mp19 for sequencing the gene coding for the yeast ubiquitinhydrolase.

FIG. 5 (parts A-D) depicts the nucleotide sequence and predicted aminoacid sequence of the yeast ubiquitin hydrolase YUH-1 as shown in FIG. 3and its flanking region from HindIII to BamHI. The location of the threesequenced peptide (numbered 14, 8 and 17) is indicated with stars. The53-mer probe sequence is shown below the correct DNA sequence.Mismatches are indicated as lower case letters. The probe is 87%identical with the correct DNA sequence.

FIG. 6 depicts the construction of the expression plasmid pTRP-YUH frompTRP-UbiA used to transform E. coli to overproduce ubiquitin hydrolase.

FIG. 7 depicts SDS-polyacrylamide gels of the protein products producedwhen E. coli is transformed with pTRP-YUH and grown under inducingconditions (lane a) or non-inducing conditions (lane b), and purified tohomogeneity (lane c). "H" designates the location of the band forubiquitin hydrolase (at 26 kD).

FIG. 8 depicts the construction of lambda gt11 TRP-YUH that is made frompTRP-YUH and is integrated into the genome of an E. coli strain.

FIG. 9 depicts the construction of pT7-12ST2HGH, an intermediate plasmidthat supplies the STII signal and the phi 10 promoter recognized by T7polymerase.

FIG. 10 shows the final steps in the assembly of the pT7-12ST2TPA-1vector bearing the STII-tPA gene under the transcriptional control ofthe phi 10 promoter.

FIG. 11 discloses the construction of pAPST2IFN-γΔNdeI-AvaI.

FIG. 12 depicts the construction, from pT7-12ST2TPA-1 andpAPST2IFN-γΔNdeI-AvaI, of pT7-3ST2TPA.

FIG. 13 depicts the construction, from pT7-3ST2TPA and pTRP-UbiA, ofpT7-3UbiAP, which contains the ubiquitin-relaxin A fusion synthetic DNAfragment driven by the phi 10 promoter.

FIG. 14 depicts SDS-polyacrylamide gels of the protein products producedupon in vivo cleavage of ubiquitin-relaxin A and ubiquitin-relaxin Bfusion polypeptides expressed from pT7-3UbiAP and pT7-12UbiB in an E.coli strain that has integrated into its genome lambda gt11 TRP-YUH,expressing the YUH-1 gene.

FIG. 15 depicts the construction of pCGY379.

FIG. 16 depicts the construction, from pT7-3ST2TPA and pCGY379, ofpYUH::URA3 that contains the interrupted YUH-1 gene.

FIG. 17 depicts the SalI to EcoRI 0.847 and 1.95 kb fragments for theuninterrupted and interrupted YUH-1 genes, respectively.

FIG. 18 depicts Southern blots of diploid and haploid yeast that aretransformed with the SalI-EcoRI fragment containing the YUH::URA3 geneinterruption.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions

The term "ubiquitin hydrolase" as used herein refers to an enzyme,whether having the sequence of the native molecule or being a derivativeor amino acid sequence variant thereof, that possesses ubiquitinhydrolase biological activity. Biological activity is one or both of (a)the capability to hydrolyze a ubiquitin-polypeptide conjugate at theamide bond linking the ubiquitin and polypeptide, thereby yieldingintact polypeptide with an unconjugated, mature N-terminus, or (b) theability to cross-react with an antibody that binds to the FIG. 5polypeptide. There are at least two naturally occurring yeast ubiquitinghydrolase proteins. The full-length version of one of these, whichcorresponds to the one with the amino acid sequence of FIG. 5, has amolecular weight of about 29,000 daltons on a reducing SDS-PAGE gel. Theamino acid sequence from the cloned gene revealed the molecular weightas 26,000 daltons. Generally, the enzyme is from eukaryotes.

Derivatives and amino acid sequence variants are defined as molecules inwhich the amino acid sequence, or other feature of a native ubiquitinhydrolase has been modified covalently or noncovalently. Amino acidsequence yeast variants include not only alleles of the FIG. 5 sequence,but also predetermined mutations thereof. Generally, amino acid sequencevariants have an amino acid sequence with at least about 80% homology,and more typically at least about 90% homology, to that of a nativeubiquitin hydrolase, e.g., the one shown in FIG. 5. Henceforth, the term"ubiquitin hydrolase" shall mean any one of the native sequences or avariant form unless otherwise appropriate.

Thus, included within the scope of the present invention is a yeastubiquitin hydrolase having the amino acid sequence as set forth in FIG.5 (YUH-1), analogous ubiquitin hydrolase proteins from other microbial,vertebrate, or invertebrate eukaryotes species such as insect, human,bovine, equine, porcine, ovine, canine, murine, feline ubiquitinhydrolase, and the like, a yeast ubiquitin hydrolase (designated hereinas YUH-2) encoded by DNA that does not hybridize with the nucleotidesequence shown in FIG. 5, even under low stringency, and biologicallyactive amino acid sequence variants of these ubiquitin hydrolases,including alleles and in vitro-generated covalent derivatives ofubiquitin hydrolase proteins that demonstrate the enzyme's activity.

The term "polypeptide" refers to a product with more than one peptidebond, including a dipeptide, tripeptide, and proteins of any size, or amutant or fragment thereof.

The term "ubiquitin-polypeptide conjugate" refers to conjugates ofubiquitin (where the 76th amino acid is glycine) to the polypeptidedefined above at the C-terminus of the ubiquitin, where the polypeptidehas as its N-terminal residue any amino acid except proline.

The expression "at least 70% homogeneity" refers to the weight of aubiquitin hydrolase in total protein, as determined by a comparativevisual inspection of a silver-stained SDS-PAGE gel for relativeintensities of the bands.

The term "buffer" refers to a buffer that is characterized by itsability to stabilize the enzyme herein at a suitable pH range, ofgenerally around 3 to 10, more preferably 4 to 8.

B. Modes of Carrying Out the Invention 1. Purification of A UbiquitinHydrolase

The steps involved in purifying a ubiquitin hydrolase of this inventionare enumerated below. This method is useful for purifying a ubiquitinhydrolase from recombinant or non-recombinant cells.

Eukaryotic cells, preferably yeast such as Saccharomyces cerevisiae oranother yeast strain, are fermented, as by using standard conditionsknown in the art, and a fermentation paste is obtained. The paste ishomogenized and the portion from the homogenate containingubiquitin-hydrolase activity is recovered, preferably by centrifugation.The activity may be assayed as described in the examples.

A precipitate containing ubiquitin-hydrolase activity is salted out fromthe recovered hydrolase-containing portion. Preferably the salting outis done using ammonium sulfate fractionation, but any salt suitable forthis purpose may be employed.

A solution of the precipitate is contacted with an ion exchange resinand the ubiquitin-hydrolase-active fraction is recovered. Preferably theion exchange resin is a DEAE chromatography column and the fraction isadsorbed to the column and recovered from the column.

The ubiquitin hydrolase-active fraction is contacted with a hydrophobicaffinity resin, such as a phenyl, octyl, or cetyl sepharosechromatographic column, and the ubiquitin-hydrolase-active fractionadsorbed to the resin is obtained and recovered. The fraction recoveredfrom this step is preferably dialyzed against a buffer before the nextstep is performed.

The ubiquitin hydrolase-active fraction is contacted with an adsorptionchromatography resin, such as a hydroxyapatite or silica column, and theubiquitin-hydrolase-active fraction adsorbed to the resin is recovered.The fraction recovered from this step is preferably dialyzed against abuffer before the next step is performed. Most preferably, theadsorption chromatography is done by hydroxyapatite columnchromatography and the active fraction is adsorbed to the column andrecovered therefrom.

The ubiquitin-hydrolase-active fraction is again contacted with an ionexchange resin, such as a DEAE chromatography column, and thehydrolase-active fraction is adsorbed to the column and recoveredtherefrom.

The ubiquitin hydrolase is then generally isolated from theubiquitin-hydrolase-active fraction in a purity of at least 70% by theweight of the total protein. Liquid chromatography may be employed inthis isolation procedure.

2. Modifications of Ubiquitin Hydrolases

Derivatives and amino acid sequence variants of ubiquitin hydrolases areuseful for their enzymatic activity, as is set forth elsewhere herein,as well as for their ability to bind to antiubiquitin hydrolaseantibodies. The derivatives and variants possessing the lattercharacteristic are useful in purifying antibodies or, when labeled, asreagents in immunoassays for ubiquitin hydrolase, whether or not suchderivatives and variants retain their enzymatic activity.

a. Covalent modification

Covalent modifications of a ubiquitin hydrolase molecule are includedwithin the scope of this invention. Variant ubiquitin hydrolasefragments having up to about 100 residues may be conveniently preparedby in vitro synthesis. Such modifications may be introduced into themolecule by reacting targeted amino acid residues of the purified orcrude protein with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. The resultingcovalent derivatives are useful in programs directed at identifyingresidues important for biological activity.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3- diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ¹²⁵ I or ¹³¹ I toprepare labeled proteins for use in radioimmunoassay, the chloramine Tmethod described above being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R'-N-C-N-R') such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking theubiquitin hydrolase to a water-insoluble support matrix or surface foruse in the method for cleaving ubiquitin fusion polypeptides to releaseand recover the cleaved polypeptide. Commonly used crosslinking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl group.

b. Mutation(s) in the DNA

Amino acid sequence variants of a ubiquitin hydrolase can also beprepared by mutations in the DNA. Such variants include, for example,deletions from, or insertions or substitutions of, residues within theamino acid sequence shown in FIG. 3. Any combination of deletion,insertion, and substitution may also be made to arrive at the finalconstruct, provided that the final construct possesses the desiredactivity. Obviously, the mutations that will be made in the DNA encodingthe variant must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure (see EP 75,444A).

At the genetic level, these variants ordinarily are prepared bysite-directed mutagenesis of nucleotides in the DNA encoding theubiquitin hydrolase, thereby producing DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture. The variantstypically exhibit the same qualitative biological activity as thenaturally occurring analog.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed ubiquitin hydrolase variants screened for the optimalcombination of desired activity. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example, site-specific mutagenesis.

Preparation of ubiquitin hydrolase variants in accordance herewith ispreferably achieved by site-specific mutagenesis of DNA that encodes anearlier prepared variant or a nonvariant version of the protein.Site-specific mutagenesis allows the production of ubiquitin hydrolasevariants through the use of specific oligonucleotide sequences thatencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the deletion junction being traversed. Typically, a primer ofabout 20 to 25 nucleotides in length is preferred, with about 5 to 10residues on both sides of the junction of the sequence being altered. Ingeneral, the technique of site-specific mutagenesis is well known in theart, as exemplified by publications such as Adelman et al., DNA, 2: 183(1983), the disclosure of which is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily commercially available and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phase origin of replication (Veira et al., Meth.Enzymol., 153: '3 [1987]) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant protein. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA), 75: 5765 (1978). This primer is thenannealed with the single-stranded protein-sequence-containing vector,and subjected to DNA-polymerizing enzymes such as E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatecells such as JM101 cells and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

After such a clone is selected, the mutated protein region may beremoved and placed in an appropriate vector for protein production,generally an expression vector of the type that may be employed fortransformation of an appropriate host.

c. Types of Mutations

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably 1 to 10 residues, and typically arecontiguous.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions of from one residue to polypeptides of essentially unrestrictedlength, as well as intrasequence insertions of single or multiple aminoacid residues. Intrasequence insertions (i.e., insertions within themature ubiquitin hydrolase sequence) may range generally from about 1 to10 residues, more preferably 1 to 5. An example of a terminal insertionincludes a fusion of a signal sequence, whether heterologous orhomologous to the host cell, to the N-terminus of the ubiquitinhydrolase to facilitate the secretion of mature ubiquitin hydrolase fromrecombinant hosts.

The third group of variants are those in which at least one amino acidresidue in the ubiquitin hydrolase molecule, and preferably only one,has been removed and a different residue inserted in its place. Suchsubstitutions preferably are made in accordance with the following Table1 when it is desired to modulate finely the characteristics of aubiquitin hydrolase molecule.

                  TABLE 1                                                         ______________________________________                                        Original Residue Exemplary Substitutions                                      ______________________________________                                        Ala              gly; ser                                                     Arg              lys                                                          Asn              gln; his                                                     Asp              glu                                                          Cys              ser                                                          Gln              asn                                                          Glu              asp                                                          Gly              ala; pro                                                     His              asn; gln                                                     Ile              leu; val                                                     Leu              ile; val                                                     Lys              arg; gln; glu                                                Met              leu; tyr; ile                                                Phe              met; leu; tyr                                                Ser              thr                                                          Thr              ser                                                          Trp              tyr                                                          Tyr              trp; phe                                                     Val              ile; leu                                                     ______________________________________                                    

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions thatin general are expected to produce the greatest changes in ubiquitinhydrolase properties will be those in which (a) glycine and/or prolineis substituted by another amino acid or is deleted or inserted; (b) ahydrophilic residue, e.g., seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, oralanyl; (c) a cysteine residue is substituted for (or by) any otherresidue; (d) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) a residue havingan electronegative charge, e.g., glutamyl or aspartyl; or (e) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having such a side chain, e.g., glycine.

Most deletions and insertions, and substitutions in particular, are notexpected to produce radical changes in the characteristics of theubiquitin hydrolase molecule. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. For example, a varianttypically is made by site-specific mutagenesis of the native ubiquitinhydrolase-encoding nucleic acid, expression of the variant nucleic acidin recombinant cell culture, and, optionally, purification from the cellculture, for example, by immunoaffinity absorption on a rabbitpolyclonal anti-ubiquitin hydrolase column (to absorb the variant bybinding it to at least one remaining immune epitope).

The activity of the cell lysate or purified ubiquitin hydrolase variantis then screened in a suitable screening assay for the desiredcharacteristic. For example, a change in the immunological character ofthe ubiquitin hydrolase, such as affinity for a given antibody, ismeasured by a competitive-type immunoassay. Changes in immunomodulatoractivity are measured by the appropriate assay. Modifications of suchprotein properties as redox or thermal stability, hydrophobicity,susceptibility to proteolytic degradation, or the tendency to aggregatewith carriers or into multimers are assayed by methods well known to theordinarily skilled artisan.

3. Recombinant Expression

The ubiquitin hydrolase molecule desired may be prepared by anytechnique, including recombinant methods. Likewise, an isolated DNA isunderstood herein to mean chemically synthesized DNA, cDNA, chromosomal,or extrachromosomal DNA with or without the 3'- and/or 5'-flankingregions. Preferably, the desired ubiquitin hydrolase herein is made bysynthesis in recombinant cell culture.

For such synthesis, it is first necessary to secure nucleic acid thatencodes a ubiquitin hydrolase. DNA encoding a ubiquitin hydrolasemolecule may be obtained from yeast or other sources than yeast by (a)obtaining a DNA library from the appropriate strain, (b) conductinghybridization analysis with labeled DNA encoding the ubiquitin hydrolaseor fragments thereof (up to or more than 100 base pairs in length) todetect clones in the library containing homologous sequences, and (c)analyzing the clones by restriction enzyme analysis and nucleic acidsequencing to identify full-length clones. DNA that is capable ofhybridizing to a ubiquitin-hydrolase-encoding DNA under stringentconditions is useful for identifying DNA encoding the particularubiquitin hydrolase desired. Stringent conditions are defined furtherbelow. If full-length clones are not present in a cDNA library, thenappropriate fragments may be recovered from the various clones using thenucleic acid sequence information disclosed herein for the first timeand ligated at restriction sites common to the clones to assemble afull-length clone encoding the ubiquitin hydrolase. Alternatively,genomic libraries will provide the desired DNA. The sequence of theyeast DNA encoding one type of yeast ubiquitin hydrolase that wasultimately determined is shown in FIG. 5.

Once this DNA has been identified and isolated from the library it isligated into a replicable vector for further cloning or for expression.

In one example of a recombinant expression system a ubiquitin hydrolaseis expressed in prokaryotes by transforming with an expression vectorcomprising DNA encoding the ubiquitin hydrolase. It is preferable totransform host cells capable of accomplishing such processing so as toobtain the hydrolase in the culture medium or periplasm of the hostcell.

a. Useful Host Cells and Vectors

The vectors and methods disclosed herein are suitable for use in hostcells over a wide range of prokaryotic and eukaryotic organisms.

In general, of course, prokaryotes are preferred for the initial cloningof DNA sequences and construction of the vectors useful in theinvention. For example, E. coli K12 strain MM 294 (ATCC No. 31,446) isparticularly useful. Other microbial strains that may be used include E.coli strains such as E. coli B and E. coli X1776 (ATCC No. 31,537).These examples are, of course, intended to be illustrative rather thanlimiting.

Prokaryotes may also be used for expression. The aforementioned strains,as well as E. coli strains W3110 (F-, lambda-, prototrophic, ATCC No.27,325), K5772 (ATCC No. 53,635), and SR101, bacilli such as Bacillussubtilis, and other enterobacteriaceae such as Salmonella typhimurium orSerratia marcesans, and various pseudomonas species, may be used.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences that are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., Gene, 2: 95 [1977]). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid or phage, must also contain, or be modified tocontain, promoters that can be used by the microbial organism forexpression of its own proteins.

Those promoters most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase) and lactose promoter systems(Chang et al., Nature, 375: 615 [1978]; Itakura et al., Science, 198:1056 [1977]; Goeddel et al., Nature, 281: 544 [1979]) and a tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res., 8: 4057[1980]; EPO Appl. Publ. No. 0036,776). While these are the most commonlyused, other microbial promoters have been discovered and utilized, anddetails concerning their nucleotide sequences have been published,enabling a skilled worker to ligate them functionally with plasmidvectors (see, e.g., Siebenlist et al., Cell, 20: 269 [1980]).

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures,may also be used. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example (Stinchcomb et al., Nature,282: 39 [1979]; Kingsman et al., Gene, 7: 141 [1979]; Tschemper et al.,Gene, 10: 157 [1980]), is commonly used. This plasmid already containsthe trp1 gene that provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example, ATCC No.44,076 or PEP4-1 (Jones, Genetics, 85: 12 [1977]). The presence of thetrp1 lesion as a characteristic of the yeast host cell genome thenprovides an effective environment for detecting transformation by growthin the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073[1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.,7: 149 [1968]; Holland et al., Biochemistry, 17: 4900 [1978]), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3' of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions, are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years [Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)]. Examples of such useful host cell linesare VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, andW138, BHK, COS-7, 293, and MDCK cell lines. Expression vectors for suchcells ordinarily include (if necessary) an origin of replication, apromoter located in front of the gene to be expressed, along with anynecessary ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment that also contains the SV40 viral origin of replication (Fierset al., Nature, 273: 113 [1978]). Smaller or larger SV40 fragments mayalso be used, provided there is included the approximately 250-bpsequence extending from the HindIII site toward the BglI site located inthe viral origin of replication. Further, it is also possible, and oftendesirable, to utilize promoter or control sequences normally associatedwith the desired gene sequence, provided such control sequences arecompatible with the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

Satisfactory amounts of protein are produced by cell cultures; however,refinements, using a secondary coding sequence, serve to enhanceproduction levels even further. One secondary coding sequence comprisesdihydrofolate reductase (DHFR) that is affected by an externallycontrolled parameter, such as methotrexate (MTX), thus permittingcontrol of expression by control of the methotrexate concentration.

In selecting a preferred host cell for transfection by the vectors ofthe invention that comprise DNA sequences encoding both ubiquitinhydrolase and DHFR protein, it is appropriate to select the hostaccording to the type of DHFR protein employed. If wildtype DHFR proteinis employed, it is preferable to select a host cell that is deficient inDHFR, thus permitting the use of the DHFR coding sequence as a markerfor successful transfection in selective medium that lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, Proc. Natl.Acad. Sci. (USA) 77: 4216 (1980).

On the other hand, if DHFR protein with low binding affinity for MTX isused as the controlling sequence, it is not necessary to useDHFR-deficient cells. Because the mutant DHFR is resistant tomethotrexate, MTX-containing media can be used as a means of selectionprovided that the host cells are themselves methotrexate sensitive. Mosteukaryotic cells that are capable of absorbing MTX appear to bemethotrexate sensitive. One such useful cell line is a CHO line, CHO-K1(ATCC No. CCL 61).

b. Typical Methodology Employable

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to prepare the plasmids required.

If blunt ends are required, the preparation may be treated for 15minutes at 15° C. with 10 units of Polymerase I (Klenow),phenol-chloroform extracted, and ethanol precipitated.

Size separation of the cleaved fragments may be performed using 6percent polyacrylamide gel described by Goeddel et al., Nucleic AcidsRes., 8: 4057 (1980).

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are typically used to transform E. coli K12 strain 294(ATCC 31,446) or other suitable E. coli strains, and successfultransformants selected by ampicillin or tetracycline resistance whereappropriate. Plasmids from the transformants are prepared and analyzedby restriction mapping and/or DNA sequencing by the method of Messing etal., Nucleic Acids Res., 9: 309 (1981) or by the method of Maxam et al.,Methods of Enzymology, 65: 499 (1980).

After introduction of the DNA into the mammalian cell host and selectionin medium for stable transfectants, amplification of DHFR-protein-codingsequences is effected by growing host cell cultures in the presence ofapproximately 20,000-500,000 nM concentrations of methotrexate, acompetitive inhibitor of DHFR activity. The effective range ofconcentration is highly dependent, of course, upon the nature of theDHFR gene and the characteristics of the host. Clearly, generallydefined upper and lower limits cannot be ascertained. Suitableconcentrations of other folic acid analogs or other compounds thatinhibit DHFR could also be used. MTX itself is, however, convenient,readily available, and effective.

Other techniques employable are described in a section just prior to theexamples.

4. Process for Cleaving Fusion Polypeptides in vitro

The ubiquitin hydrolase molecules herein are particularly useful in aprocess for readily processing in vitro a fusion polypeptide betweenubiquitin and any polypeptide product desired. Ubiquitin fusionpolypeptides are expressed generally by a chimeric gene constructcomprising a ubiquitin gene ligated at its 3' end to the 5' end of agene coding for the desired polypeptide. The ubiquitin gene is obtainedfrom a natural source and cloned into an appropriate vector, asdescribed in WO 88/02406, supra, the disclosure of which is incorporatedherein by reference, or it is synthesized chemically, using, e.g., themethod described by Ecker et al., J. Biol. Chem., 262:3524-3527 (1987)and Ecker et al., J. Biol. Chem., 262: 14213-14221 (1987), thedisclosures of which are incorporated by reference. The fusion in turnoptionally contains an N-terminal signal sequence to facilitatesecretion of the fusion polypeptide.

The codon for the N-terminal amino acid of the desired polypeptide islocated directly adjacent to the 3' end of the ubiquitin gene or isseparated by any number of nucleotide triplets (typically one, two, orthree triplets) that need not encode any particular sequence but whichkeep the gene encoding the desired polypeptide in the correct readingframe.

The desired polypeptide may be any polypeptide, including, but notlimited to, mammalian polypeptides, such as, e.g., a growth hormone,including human growth hormone, des-N-methionyl human growth hormone,and bovine growth hormone; insulin A-chain, insulin B-chain; proinsulin;factor VIII; a plasminogen activator, such as urokinase or humantissue-type plasminogen activator (t-PA); tumor necrosis factor-alphaand -beta; enkephalinase; a serum albumin such as human serum albumin;mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin associated peptide; a microbial protein,such as beta-lactamase; DNase; tissue factor protein; inhibin; activin;nerve growth factor such as NGF-β; platelet-derived growth factor;fibroblast growth factor; transforming growth factor (TGF) such asTGF-alpha and TGF-beta; insulin-like growth factor-I and -II;insulin-like growth factor binding proteins; CD-4; erythropoietin; aninterferon such as interferon-alpha, -beta, and -gamma; colonystimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins(ILs), e.g., IL-1, IL-2, IL-3, IL-4, etc.; superoxide dismutase; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; fragments of any of the above-listed polypeptides;and the like. In addition, one or more predetermined amino acid residueson the polypeptide may be substituted, inserted, or deleted, withoutadversely affecting the expression and/or ubiquitin hydrolase processingof the fusion.

Preferably, the polypeptide herein is relaxin or insulin A-chain orB-chain, prorelaxin, proinsulin, an interferon, an interleukin, a growthhormone, a nerve growth factor, a transforming growth factor, aninsulin-like growth factor, or DNase. Most preferably, the polypeptideherein is relaxin or insulin A-chain or B-chain, prorelaxin, proinsulin,interferon-gamma, or des-N-methionyl human growth hormone. Also,preferably the polypeptide is a polypeptide heterologous to the hostcell in which it is expressed, and is a human polypeptide.

The fusion is produced in a composition comprising contaminant productsof recombinant cell culture and then cleaved so as to recover thedesired polypeptide product. The polypeptide is conjugated to theC-terminus of the ubiquitin and contains any amino acid at itsN-terminus. The host cell culture containing the fusion is grown inculture medium appropriate to the host and harvested by a methoddependent on whether the fusion is secreted. In general, the cells arelysed and centrifuged to spin out the cellular debris and recover thefusion protein in the supernatant. The supernatant or secreted fusionmaterial recovered from the periplasm, e.g., by osmotic shock, or fromthe extracellular medium is then contacted with a reagent havingspecific affinity for ubiquitin so that the conjugate is adsorbed on thereagent. This reagent may be any reagent, such as a cellular proteinthat interacts with the ubiquitin or an anti-ubiquitin antibody,preferably a monoclonal antibody. Most preferably, the separation takesplace on an affinity chromatography column to which a monoclonalantibody against ubiquitin is bound.

In the next step, the reagent having specific affinity for ubiquitin andits adsorbed conjugate is separated from the rest of the host cellculture, and the conjugate is recovered from the reagent. If theadsorbed conjugate is on an affinity column, the conjugate adsorbed tothe antibody is recovered by elution from the column using a pH gradientof 4-5.

In the following step, the recovered conjugate is contacted with aubiquitin hydrolase, whereby the conjugate is hydrolyzed to ubiquitinand mature polypeptide and the hydrolase is immobilized. This may beaccomplished by passing the eluted conjugate through a column to whichthe ubiquitin hydrolase is bound. Alternatively, the conjugate iscontacted with the ubiquitin hydrolase and then an antibody against thehydrolase that is immobilized is used to separate the hydrolase from theconjugate.

The recovered material is then contacted with a reagent having specificaffinity for ubiquitin so that any residual conjugate and free ubiquitinare adsorbed on the reagent, and the polypeptide is recovered free fromthe reagent and the materials adsorbed thereon. Again, this reagent maybe a monoclonal antibody against ubiquitin, which may be bound to anaffinity column.

For the success of this process, the host cell preferably produces noendogenous ubiquitin hydrolase that will interfere with the recoveryprocess. This can be achieved either by using a prokaryotic host, whichin general produces no ubiquitin hydrolase, or by employing deletion ortransposon mutagenesis to rid the host cell, i.e., a eukaryotic hostcell, of all genes that code for endogenous ubiquitin hydrolases. It mayalso be desirable to select host cells deficient in endogenous proteasesthat might degrade the fusion polypeptide if it is producedintracellularly, e.g., in a prokaryotic host.

In another method for recovering the cleaved polypeptide, after the hostcell culture producing the conjugate is harvested as described above,the culture is contacted with the reagent having specific affinity forubiquitin so that the conjugate is adsorbed on the reagent (the reagentbeing defined as described above). The reagent and its adsorbedconjugate are separated from the rest of the culture. Then the reagenton which is adsorbed the conjugate is contacted with the ubiquitinhydrolase. The hydrolase and polypeptide are separated from the reagent,and, in a final step, the polypeptide is separated from the hydrolase.The same preferred embodiments mentioned for the first process alsoapply to this process.

5. Antibodies to Ubiquitin

Antibodies to ubiquitin generally are raised in animals by multiplesubcutaneous or intrapertitoneal injections of ubiquitin and anadjuvant. It may be useful to conjugate the ubiquitin to a protein thatis immunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCL₂ or R¹ N=C=NR. Also,aggregating agents such as alum may be used to enhance the immuneresponse.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with three volumes of Freund's complete adjuvant andinjecting the solution intradermally at multiple sites. One month laterthe animals are boosted with 1/5 to 1/10 the original amount ofconjugate is Freund's complete adjuvant by subcutaneous injection atmultiple sites. Seven to 14 days later animals are bled and the serum isassayed for anti-ubiquitin titer. Animals are boosted until the titerplateaus. Preferably, the animal is boosted with the conjugate of thesame ubiquitin polypeptide, but conjugated to a different protein and/orthrough a different crosslinking agent.

Monoclonal antibodies are prepared by recovering spleen cells fromimmunized animals and immortalizing the cells in conventional fashion,e.g., by fusion with myeloma cells or by Epstein-Barr virustransformation and screening for clones expressing the desired antibody.Monoclonal antibodies to ubiquitin are described, for example, in Smithand Fried, Fed. Proc., 46(6): 2087 (1987), and polyclonal antisera aredescribed by Redman et al., J. Biol. Chem., 263: 4926-4931 (1988).

6. Process for Cleaving Fusion Polypeptides in vivo

Ubiquitin hydrolases are also particularly useful in a method forprocessing in vivo a fusion polypeptide between ubiquitin and anypolypeptide product desired. The gene construct for the conjugate is asdescribed in Section 4 above.

This gene construct is transformed into any prokaryotic host cells thathave had integrated into their genome the gene coding for a ubiquitinhydrolase, preferably as a single copy in the chromosome. Such cells maybe prepared by cloning a DNA construct having a promoter linked to the5' end of a ubiquitin hydrolase gene into a lambda phage and integratingit into the appropriate strain. Preferably the host cells are E. colicells, e.g., those having the characteristics of E. coli strain K5808,which was deposited in the American Type Culture Collection, 12301Parklawn Drive, Bethesda, Md., USA under ATCC Accession No. 53,832 onNov. 30, 1988. This strain contains the YUH-1 gene construct driven bythe trp promoter in a λgt11 phage integrated into the lambda attachmentsite between gal and bio of E. coli strain K5772.

Strain K5772 (ATCC No. 53,635) contains the T7 RNA polymerase geneinserted into the chromosomal lacZ operon that is thus inducible byaddition of isopropylthiogalactoside (IPTG) to the media. Theconstitutive level of expression of T7 RNA polymerase depends on theuntranslated domain located 5' to the polymerase structural gene. The T7RNA polymerase gene integrated in E. coli K5772 contains the followingsequence (the complementary strand is not shown): ##STR1## The HpaI siteis the first HpaI of the E. coli lacZ.

The resulting transformed host cells are cultured so as to express theubiquitin-polypeptide conjugate. Culturing is preferably accomplished byinducing the promoter for the hydrolase gene. Thus, if trp is used asthe promoter, the cells are cultured in minimal media withouttryptophan. The induction results in efficient cleavage ofubiquitin-relaxin A-chain fusion in 20 minutes. As soon as the conjugateis expressed, it is cleaved by the ubiquitin hydrolase coded for in thegenome.

7. Kit Components

A composition of the ubiquitin hydrolase herein may be formulated in abuffer for stability purposes. The buffer can be composed of inorganicor organic salts and includes, for example, citrate, phosphate, or Trisbuffer, depending on the pH desired.

Further, the composition of the ubiquitin hydrolase may be one componentof a kit, which also contains an immobilized antibody to a ubiquitinhydrolase as the second component. The antibody may be immobilized asdescribed above regarding modifications to ubiquitin. Such a kit can beused for performing cleavage of fusion proteins containing ubiquitinconjugated to the desired protein.

In other to simplify the examples and claims, certain frequentlyoccurring methods will be referenced by shorthand phrases.

"Transfection" refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described by Cohen, S. N. Proc. Natl.Acad. Sci. (USA), 69: 2110 (1972) and Mandel et al., J. Mol. Biol,53:154 (1970), is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham, F. andvan der Eb, A., Virology, 52: 456-457 (1978) is preferred. Generalaspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983.Transformations into yeast are typically carried out according to themethod of Van Solingen, P., et al., J. Bact., 130: 946 (1977) and Hsiao,C. L., et al., Proc. Natl. Acad. Sci. (USA) 76: 3829 (1979). However,other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used.

As used herein, the expression "hybridize under stringent conditions" todescribe certain DNA sequences encompassed within the scope of thisinvention refers to hybridizing under conditions of low ionic strengthand high temperature for washing, for example, 0.15M NaCl/0.015M sodiumcitrate/0.1% NaDodSO₄ at 50° C., or alternatively the presence ofdenaturing agents such as formamide, for example, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate, at 42° C. for hybridization. "Hybridizeunder low stringency" refers to hybridizing under conditions of 20%formamide, 5 X SSPE, 42° C.

"Site-directed mutagenesis" is a technique standard in the art, and isconducted using a synthetic oligonucleotide primer complementary to asingle-stranded phage DNA to be mutagenized except for limitedmismatching, representing the desired mutation. Briefly, the syntheticoligonucleotide is used as a primer to direct synthesis of a strandcomplementary to the phage, and the resulting double-stranded DNA istransformed into a phage-supporting host bacterium. Cultures of thetransformed bacteria are plated in top agar, permitting plaque formationfrom single cells that harbor the phage. Theoretically, 50% of the newplaques will contain the phage having, as a single strand, the mutatedform; 50% will have the original sequence. The plaques are hybridizedwith kinased synthetic primer at a temperature that permitshybridization of an exact match, but at which the mismatches with theoriginal strand are sufficient to prevent hybridization. Plaques thathybridize with the probe are then selected and cultured, and the DNA isrecovered.

"Operably linked" refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence "operablylinked" to control sequences refers to a configuration wherein thecoding sequence can be expressed under the control of these sequencesand wherein the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, then synthetic oligonucleotide adaptors orlinkers are used in accord with conventional practice.

"Control sequences" refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, a ribosome bindingsite, and possibly, other as yet poorly understood sequences. Eukaryoticcells are known to utilize promoters, polyadenylation signals, andenhancers.

"Expression system" refers to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. To effect transformation, the expression system may beincluded on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

As used herein, "cell," "cell line," and "cell culture" are usedinterchangeably and all such designations include progeny. Thus,"transformants" or "transformed cells" includes the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Mutant progeny that have the same functionality as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the sites for which each is specific iscalled a restriction site. The various restriction enzymes used hereinare commercially available and their reaction conditions, cofactors, andother requirements as established by the enzyme suppliers are used.Restriction enzymes commonly are designated by abbreviations composed ofa capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1μg of plasmid or DNA fragment is used with about 1-2 units of enzyme inabout 20 μl of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation of about 1 hour at 37° C. is ordinarily used,but may vary in accordance with the supplier's instructions. Afterincubation, protein is removed by extraction with phenol and chloroform,and the digested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5' phosphates to prevent the two restriction cleavedends of a DNA fragment from "circularizing" or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5' terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional (T. Maniatis et al., 1982, MolecularClonging: A Laboratory Manual (New York: Cold Spring Harbor Laboratory,1982) pp. 133-134).

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby elctrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see R. Lawn et al., Nucleic Acids Res.9:6103-6114 (1981), and d. Goeddel et al., Nucleic Acids Res. 8:4057(1980).

"Southern Analysis" is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labelled oligonucleotide or DNA fragment. For the purposesherein, unless otherwise provided, Southern analysis shall meanseparation of digests on 1 percent agarose, denaturation, and transferto nitrocellulose by the method of E. Southern, J. Mol. Boil. 98:503-517 (1975), and hybridization as described by T. Maniatis et al.,Cell 15: 687-701 (1978).

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double-stranded nucleic acid fragments (T. Maniatis et al., 1982,supra, p. 146). Unless otherwise provided, ligation may be accomplishedusing known buffers and conditions with 10 units of T4 DNA ligase("ligase") per 0.5 μg of approximately equimolar amounts of the DNAfragments to be ligated.

"Preparation" of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofManiatis et al., 1982, supra, p. 90, may be used.

"Oligonucleotides" are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP Pat. Pub. No. 266,032published May 4, 1988, or via deoxynucleoside H-phosphonateintermediates as described by Froehler et al., Nucl. Acids Res., 14:5399-5407 [1986]). They are then purified on polyacrylamide gels.

The following examples are intended to illustrate the best mode nowknown for practicing the invention, but the invention is not to beconsidered limited thereto.

All literature citations herein are expressly incorporated by reference.

EXAMPLE I I. Protein Assay

A. Preparation of 35-S labeled protein substrate for ubiquitin hydrolase

This labeling procedure was carried out in vivo in an E. coli strainthat contains an integrated bacteriophage T7 RNA polymerase gene and aplasmid containing a gene for a protein substrate to be labeled. Theexpression of the protein substrate gene was under the control of T7 RNApolymerase promoter, whose sequences were located 5' to the proteinsubstrate gene on the plasmid. The expression of T7 RNA polymerase genewas under the control of the lac promoter of E. coli.

DNA fragments encoding yeast ubiquitin fusion polypeptides weresynthesized chemically on a DNA synthesizer using the method of Froehleret al., supra. The synthetic ubiquitin fusion gene for human (H2)relaxin B-chain of 32 amino acids (missing the N-terminal amino acid ofrelaxin B-chain, but adding six amino acids at the junction of ubiquitinand the relaxin chain) is shown in FIG. 1a extending from the stickyends of XbaI to HindIII. It can be seen from FIG. 1a that the ubiquitingene has a convenient unique DraIII site at nucleotides 211-216 that canbe used to attach DNA encoding various proteins, if the DNA encoding theprotein to be attached is linked to the remainder of the nucleotidesneeded to construct the ubiquitin 3' end and has a DraIII site inserted.This DraIII site was used to synthesize the remaining fusion peptideswith N-terminal-truncated ubiquitin.

Thus, the truncated synthetic ubiquitin fusion gene for human (H2)relaxin B-chain of 33 amino acids is shown in FIG. 1b extending from theinternal DraIII site (sticky end) of ubiquitin to a HindIII site (stickyend) at the end of the relaxin chain. Similarly, the truncated gene forubiquitin with a gene for a cysteine dipeptide at its 3' end wasconstructed extending from the ubiquitin sticky end DraIII site to asticky end HindIII site at the 3' end of the fragment. Also, thetruncated gene for ubiquitin fused at its 3' end to the gene for human(H2) relaxin B-chain with 29 amino acids (truncated at its 3' end byfour amino acids) was synthesized in the same way, from the internalsticky end DraIII site of ubiquitin to a sticky end HindIII site at the3' end of the fragment. Finally, the truncated gene for ubiquitin fusedat its 3' end to the gene for human (H2) relaxin A-chain with 24 aminoacids was synthesized in the same way, from the internal sticky endDraIII site of ubiquitin to a sticky end HindIII site at the 3' end ofthe fragment. The nucleotide sequence of human relaxin (H2) A-chain canbe found in European Pat. Pub. No. 112,149 published Jun. 27, 1984.

FIGS. 1c and 1d show the construction of the vectors encoding theubiquitin protein substrates with the various synthetic relaxin chains.First, plasmid pT7-2 was obtained from United States BiochemicalCorporation. The plasmid DNA was cleaved with PvuI and religated. TheDNA was used to transform competent bacteria and clones were screenedfor inversion of the PvuI fragment. One such inverted clone was calledpT7-12. The purpose of this construction was to prevent high-levelexpression of the beta-lactamase gene, which otherwise would betranscribed under the control of the phi 10 promoter. pT7-12 was cleavedwith HincII and BamHI and the large fragment isolated.

A second plasmid, pTrpXAPTNF, was prepared from pBR322 and contains thetumor necrosis factor (TNF)-encoding gene under the control of the trppromoter. The construction of this gene is described fully in EP Pub.No. 168,214, published Jan. 15, 1986, the disclosure of which isincorporated herein by reference. This plasmid was cleaved with HpaI andBamHI and the small fragment was isolated. This small fragment was thenligated to the large fragment from PT7-12, to form the plasmidpT7-12TNF, which contains the TNF-encoding gene and the XbaI site withinthe trp leader ribosome binding site. The construction of pT7-12TNF isdepicted in FIG. 1c.

The plasmid pSRCex16 (described by J. P. McGrath and A. D. Levinson,Nature, 295: 423-425 [1982]) was cleaved with XbaI and HindIII and thelarge fragment isolated. The synthetic DNA shown in FIG. 1a (UbsRlxnB32)was ligated with this large fragment to yield the plasmidpTrpUbsRlxnB32, the construction of which is shown in FIG. 1d. Thisplasmid was cut with XbaI and BamHI and the small fragment was isolated.The plasmid pT7-12TNF was cleaved with XbaI and BamHI and the largefragment was isolated and ligated to the small fragment frompTrpUbsRlxnB32. The resulting plasmid was pT7-12UbsRlxnB32, wherein thesynthetic DNA fragment is under the control of the pT7 promoter.

The other plasmids, pT7-12Ubs-X, where X is for the cys-cys dipeptide,relaxin B 29 chain, relaxin B 33 chain, or relaxin A 24 chain, wereprepared as shown in FIG. 1d by cleaving the ptrpUbsRlxnB32 vector withDraIII and HindIII, isolating the large fragment and ligating it to oneof the four synthetic DraIII-HindIII fragments mentioned above (calledUbs-X) to yield the ptrpUbs-X plasmids, where X is defined above,cleaving these plasmids with XbaI and BamHI, isolating the smallfragments, and ligating them to the isolated large fragment of the XbaIand BamHI-cleaved pT7-12TNF plasmid.

Alternatively, the yeast ubiquitin gene (Ozkaynak et al., The EMBO J.,6: 1429-1439 [1987]) was assembled from eight oligonucleotide fragmentsranging from 55 to 62 nucleotides in length, with the sequence and orderas follows: ##STR2##

About 30 ng of each oligonucleotide was phosphorylated and ligatedtogether in a single reaction mixture containing 50 mM Tris-HCl (pH8.0), 10 mM MgCl₂, 0.5 mM ATP, 10 units of T4 polynucleotide kinase, and1000 units of T4 DNA ligase. The resultant DNA duplex was fractionatedin a 6% polyacrylamide gel and the DNA band corresponding to the 240base-pair fragment was excised and electroeluted.

The eluted DNA was extracted with chloroform, precipitated with ethanol,ligated with linkers so as to have XbaI and EcoRI ends, and ligated tothe large fragment isolated from a pUC-12 vector (Boehringer MannheimBiochemicals) cleaved by XbaI and EcoRI. The sequence of the ubiquitinDNA insert was verified by dideoxynucleotide DNA sequencing analysis.

Similarly, DNA corresponding to the artificial relaxin B-chain geneencoding 38 amino acids was assembled from four synthetic DNA fragmentsranging from 57 to 62 nucleotides in length (sequences given below),ligated to EcoRI and HindIII linkers, and then ligated to the largefragment isolated from EcoRI and HindIII cleaved pUC-12, and thesequence was verified. These two DNA inserts were excised from theirrespective plasmids by cleaving with XbaI and EcoRI for the firstvector, and with EcoRI and HindIII for the second vector. These twoinserts were then ligated together at the common EcoRI sites to yield aXbaI-HindIII fragment. This fragment was ligated with the large fragmentisolated from pSRCex16, cleaved by XbaI and HindIII. Synthetic DNASequences Used: ##STR3##

The fusion protein of ubiquitin and relaxin B-chain was prepared bysubstituting the synthetic DNA sequence existing between the DraIII andHindIII sites of pSRCex16 with the relevant synthetic DNA fragment sothat the ubiquitin-encoding gene was 5' to the gene for the polypeptide.The resulting hybrid gene was excised from the pSRCex16-derived plasmidby digestion with XbaI and BamHI, isolated, and inserted into the largefragment isolated from XbaI- and BamHI-cleaved pT7-12TNF.

E. coli K5772 bacteria (deposited in the American Type CultureCollection under accession number 53,635 and containing the T7 RNApolymerase in the E. coli lacZ gene) were made competent and transformedwith pT7-12UbsRlxnB32 or one of the pT7-12Ubs-X plasmids, separately.Cells were selected for resistance to carbenicillin.

Each E. coli transformant was grown overnight at 37° C. to saturation in5 ml of M9 minimal media supplemented with 50 μg/ml each of all aminoacids except for cysteine and methionine. (Because of the fastinterconversion between cysteine and methionine in vivo, labeling donein the absence of exogenously added methionine will result in thelabeling of the methionine residue in the protein. Because of theinterconversion, labeling done in the presence of exogenously addedmethionine will result in the almost nondetectable incorporation of S35into the methionine residue.) The presence of glucose ensured cataboliterepression of the lac promoter controlling T7 RNA polymerasetranscription. Fifty μg/ml of carbenicillin was also included tomaintain the stability of the plasmid.

The overnight culture was diluted 50-fold into 1 ml of M9 minimal mediasupplemented with 10 μg/ml of each amino acid except for cysteine andmethionine plus 50 μg/ml of carbenicillin.

After three hours of shaking at 37° C., isopropylthio-β-D-galactoside(IPTG) was added to the culture to give a final concentration of 1 mM toinduce the synthesis of T7 RNA polymerase. After another 30 minutes, 20mg/ml rifampicin was added to give a final concentration of 200 μg/ml toinhibit host RNA polymerase activity. Another 30 minutes later, 35Scysteine (600 Ci/mmole) was added to the culture at the ratio of 0.25mCi/ml culture to label the proteins or the cys-cys dipeptide.

The labeling was stopped by quenching the culture with cysteine at afinal concentration of 50 μg/ml and the bacterial pellet was collectedby centrifugation.

The lysis of the cell can be carried out differently depending on thepurpose of the cell lysate to be used. The following procedure was usedto prepare lysate that contains all soluble E. coli proteins (itcontains the labeled protein if it is soluble) in a solution that iscompatible with almost all enzymatic processes to be examined underaqueous conditions without further treatment. The ratio of variousreagents to culture volume was based on a 1-ml labeling culture.

To the collected bacterial pellet, 80 μl of a solution containing 50 mMTris-Cl (pH 8), 25% (w/v) sucrose was added to resuspend the cells. Thisstep as well as all the following steps were carried out at roomtemperature.

To the cell suspension, 20 μl of 5 mg/ml of egg white lysozyme freshlydissolved in 0.25M Tris-Cl (pH 8.0) was added and incubated for fiveminutes. Forty μl of 0.25M EDTA (pH 8) was then added to the suspension.

After another five minutes of incubation, cells were lysed by adding 60μl of the lytic mix containing 50 mM Tris-Cl (pH 8), 50 mM EDTA (pH 8),0.2% (v/v) NP40. It required between 5 and 10 minutes to obtain completecell lysis. The lysate was clarified by centrifugation and was ready tobe used or could be kept at -20° C. for a long period of time.

B. Ubiquitin Hydrolase Assays 1. Fusion protein cleavage assay

All assays were carried out in Eppendorf tubes with a final volume of 25μl and incubation was at 37° C. for one hour. The reaction mix containedthe following:

50 mM Tris-Cl (pH 7.5)

1 mM EDTA (pH 8.0)

10 mM dithiotreitol

1 μl S-35 Cys labeled ubiquitin-relaxin B-chain substrate 2.5 μlubiquitin hydrolase solution

After incubation, the reaction mixtures were mixed with an equal volumeof SDS sample buffer, heated to 90° C. for 5 min. The samples were thenloaded directly onto a 15% SDS-PAGE gel, and monitored byelectrophoresis to resolve the cleaved product from uncleavedsubstrates. Detection of various labeled protein species was done bydrying the gel on a piece of Whatman No. 1 paper and exposing it toX-ray film overnight. A diagram depicting the principle of the assay isshown in FIG. 2, where RGG stands for arg-gly-gly at the carboxyterminus of ubiquitin and C stands for cysteines.

Because the labeled substrate is obtained by in vivo labeling, it islimited in quantity and with undetermined specific activity. The bestway to quantitate the amount of enzyme is to carry out the abovereaction with serially diluted enzyme solutions with the same batch ofsubstrate. The fold of dilution where the enzyme activity diminished canbe used as an expression of the relative activity of a particular enzymesolution. The dilution buffer used for this purpose was the reactioncocktail with 50 μg, ml bovine serum albumin without enzyme andsubstrate.

2. Cleavage by cleaving ubiquitin-Cys-Cys

This assay is identical to the protein cleavage assay in principleexcept with a different substrate, ubiquitin-cys-cys. The cleavedproducts, ubiquitin and cys-cys, were separated by acid precipitationinstead of SDS-PAGE because cys-cys is soluble in acid while the biggerubiquitin is not.

All assays were carried out in Eppendorf tubes with a final volume of 25μl, and incubation was at 37° C. for 20 minutes. The reaction mixcontained the following:

50 mM Tris-Cl (pH 7.5)

1 mM EDTA (pH 8.0)

10 mM dithiothreitol

50 μg/ml bovine serum albumin

1 μl S-35 Cys labeled ubiquitin-Cys-Cys substrate

2.5 μl ubiquitin hydrolase solution

After incubation, 20 μl of the reaction mixture was spotted onto a GF/Cfilter disc (2.1 cm) and immediately immersed in 10% (w/v)trichloroacetic acid (TCA) contained in a beaker over ice. Individualfilter disc was labeled beforehand with Indian ink for identificationafter the washing procedures. The beaker was swirled occasionally duringa five-minute period. The TCA solution was decanted and filters werefurther washed with 5% TCA solution. After another five minutes withoccasional swirling, the TCA solution was again decanted. Filter discswere rinsed in 95% alcohol to remove TCA and dried under a heat lamp.Individual filter discs were placed into a counting vial, filled with 5ml of counting fluid, and counted in a scintillation counter. Since theinsoluble ubiquitin was retained on the filter disc, hydrolase activitywas measured by the decrease of radioactivity retained by the filterdisc.

Because the labeled substrate was obtained by in vivo labeling, it islimited in quantity and with undetermined specific activity. Thespecific activity of the hydrolase is also unknown at the present time.Therefore, the best way to quantitate the amount of the enzyme is tocarry out the above reaction with serially diluted enzyme solutions withthe same batch of substrate. The fold of dilution where the enzymeactivity resulted in 60% retention of the original amount ofradioactivity can be used as an expression of the relative activity of aparticular enzyme solution. The dilution buffer used for this purpose isthe reaction cocktail without enzyme substrate and enzyme itself.

Because both protein fusion cleavage assay and cysteine release assayappear to detect the same enzyme, it is easiest to quantitate activityby diluting enzyme solutions to obtain about 50% hydrolysis of thesubstrate in the cysteine release assay. The relative activity of anenzyme solution was then defined as that fold of dilution necessary toobtain 50% hydrolysis under the standard assay conditions.

II. Purification and Assay of a Yeast Ubiquitin Hydrolase

All of the purification steps were conducted at room temperature, exceptfor the overnight dialysis and except for storage, which was at 4° C.

A. Fermentation

The yeast strain Saccharomyces cerevisiae is grown in a ten-literfermenter at 30° C. in 2.6% yeast nitrogen base and 1% glucose to anA₆₆₀ of 3-4. Cells are then slowly fed with glucose until the A₆₆₀reached 50-100.

B. Cell Homogenization

About one kg of the yeast strain fermentation paste was resuspended at 1g/ml in Buffer A (50 mM Tris-Cl (pH8), 1 mM EDTA, 10% (v/v) glycerol,and 10 mM 2-mercaptoethanol). The resulting suspension was mixed with0.25 g/ml of glass beads (Sigma G-8893, 106 microns and finer). Thecell-glass beads suspension was blended in a Waring blender at top speedfor several 2-3 minute-pulses for a total of ten minutes (care must betaken so that the temperature does not rise during this operation). Theefficiency of cell breakage can be monitored by measuring proteinconcentration of the supernatant after a brief centrifugation of thesuspension.

After homogenization, the suspension was centrifuged at 12,000 rpm in aSorvall GSA rotor for about 30 minutes. Pellets were collected andresuspended in Buffer A (at the original 1 g/ml ratio) and were blendedagain for about five minutes (again, in two-minute pulses). Thesupernatant was collected after the centrifugation as before andcombined with the first supernatant. The combined supernatant wasfurther clarified by centrifugation at 18,000 rpm in a Sorvall SS-34rotor for 30 minutes. The protein concentration at this stage was about10 mg/ml or 20 mg/g of original wet yeast paste.

C. Ammonium Sulfate Fractionation

Ubiquitin hydrolase activity was recovered between 33% and 63%saturation of ammonium sulfate. Ammonium sulfate was added directly tothe crude, clarified supernatant to give an initial 33% saturation andthe pH of the suspension was maintained by adding about 1 μl of 1M Trisbase per g of solid ammonium sulfate added. The supernatant of the 33%saturation was then brought to 63% saturation. About half the proteinwas removed.

D. DEAE Chromatography

Protein precipitate after 63% ammonium sulfate saturation was collectedby centrifuging the suspension from above in a GSA rotor at 12,000 rpmfor 30 minutes. Protein precipitate was resuspended in 350 ml of BufferA and dialyzed against four liters of Buffer A containing 80 mM NaCl for24 hours (one buffer change in 72 hours). The dialyzed protein solutionwas loaded onto a DEAE Sephacel column (5×21 cm) equilibrated withBuffer A containing 80 mM NaCl at a flow rate of 400 ml per hour (theflow rate was slowed to about 80 ml per hour). The column wassequentially washed with Buffer A containing 80 mM NaCl, 170 mM NaCl,230 mM NaCl, and 300 mM NaCl. The buffer volume in each wash was aboutone liter. Almost all the activities eluted in the 300 mM NaCl wash andthe column matrix, which was still heavily discolored at this point, wasdiscarded. Active fractions were pooled (about 450 ml), and weresubjected to the next column fractionation.

E. Phenyl-Sepharose Chromatography

Solid ammonium sulfate was added to the pooled DEAE fraction at theratio of ten g per 100 ml of protein solution and loaded directly onto aphenyl-sepharose column (2.5×17 cm) previously equilibrated with BufferA containing 10% (w/v) ammonium sulfate at a flow rate of about 50 mlper hour. The column was then washed with 100 ml each of Buffer Acontaining 10% (w/v) and then 5% ammonium sulfate. The enzyme wassubsequently eluted with 350 ml of a linearly decreasing gradientcomposed of Buffer A with 5% ammonium sulfate and Buffer A. Activefractions, which were located in the early 20% of the linear gradient,were pooled (total volume is about 84 ml) and concentrated byultrafiltration through an Amicon membrane. The concentrated enzymesolution (about 40 ml) was dialyzed overnight against one liter ofBuffer B (50 mM Tris-Cl (pH8), 10% (v/v) glycerol, 10 mM2-mercaptoethanol).

F. Hydroxyapatite Chromatography

The dialyzed enzyme solution was loaded onto a hydroxyapatite column(1.0×7 cm) equilibrated with Buffer B. The column was washed with 10 mlof Buffer B and then eluted with 50 ml of a linear gradient betweenBuffer B and Buffer B containing 0.5M ammonium sulfate. It was furtherwashed with 10 ml each of Buffer B containing 0.5M ammonium sulfate andthen 1M ammonium sulfate. Enzyme activities were located in the gradientand then pooled (total volume is about 8 ml) and dialyzed overnightagainst Buffer A plus 0.1M NaCl.

The overall yield at this point was about 20% of the original activitypresent in the crude lysate and the overall purification was about15,000 fold.

G. Rechromatography on DEAE Sephacel

A DEAE Sephacel column (0.7×7 cm) was loaded with dialyzed enzyme andthe column was equilibrated with Buffer A containing 0.1M NaCl and thenwashed with 5 ml of the same buffer. Enzyme activities were eluted witha 30-ml linear gradient between 0.1M NaCl and 1.0M NaCl in Buffer A.

The activity was located around 0.3M NaCl as expected and the overallyield at this point was at least 15% of the original activity present inthe crude lysate.

H. SDS-PAGE

A reducing SDS-PAGE gel of the recovered activity was prepared andstained with silver stain. Upon visual inspection of the gel andcomparison of the relative densities of the bands, it was found that theubiquitin hydrolase obtained was about 70% pure based on the weight ofthe total protein in the composition. The major protein species, withmolecular weight of about 30,000 daltons, comigrating with hydrolaseactivities throughout phenyl sepharose, hydroxyapatite and the last DEAEcolumns was confirmed by cloning of the gene to be a ubiquitin hydrolaseprotein.

I. HPLC and Sequencing

The recovered activity from the DEAE column was placed on a 4000angstrom wide-pore column (100 mm×2 mm in diameter) from Synchrom, Inc.in a Hewlett Packard C4 RP-HPLC 190M equipped with a 1040 diode arraydetector. A linear gradient was used of 100% solution A to 60% solutionB in 60 minutes, wherein solution A is 0.1% trifluoroacetic acid (TFA)in water and solution B is 0.07% TFA in 1-propanol. The flow rate was200 μl per minute at room temperature and the peaks were monitored at214 and 280 nm. All the peaks with 214 nm absorption were collected inthe buffer used for the particular assay of the hydrolase activity.

There was one positive assay from the peaks collected that had a 30 kDamolecular weight on a reducing SDS-PAGE gel. One fraction from the DEAEcolumn that was 90% enriched in the 30 kDa protein was digested byadding about 5% Lycine C (Wako) to the fraction in its elution buffer.The digestion was carried out for 24 hours at 37° C. The peptidesresulting from the digestion were separated on the C4-HPLC columnmentioned above using the same conditions as described above. Theseparated peptides were sequenced on an Applied Biosystems 470A gasphase sequencer using Edman degradation. The following amino acidsequences were obtained:

1. SDPTATDLIEQELVRVRVA

2. ENVQTFSTGQSEAPEATADTNLHYI

3. NEWAYFDIY

4. NRFDDVTTQ.

III. Cloning and Expression of the Hydrolase Gene

A synthetic DNA probe was synthesized on a DNA synthesizer using themethod of Froehler et al., supra. This probe (Probe 1, 53 mer) had thefollowing sequence: ##STR4##

Total genomic DNA was isolated from a yeast strain S1799D (αtrp5 his4ade6 gal2) of Saccharomyces cerevisiae (Yeast Genetic Stock Center,Berkeley, Calif.) by the method of Smith et al., Method Enzymol., 12:538-541 (1967). 370 μg of yeast DNA was partially digested with 2.5units of Sau3AI (New England Biolabs) in 2 ml of reaction mixture.Aliquots were removed at 10, 20, and 30 min., chilled, and inactivatedwith 20 mM EDTA. The pooled, phenol-extracted DNA was fractionated bycentrifugation in 10-50% sucrose gradients in 1M NaCl, 20 mM Tris-HCl(pH 8.0), and 10 mM EDTA. The Sau3AI 10-15-kb fragments (determined byagarose gel electrophoresis) were isolated and ligated intobacteriophage λ Charon 30 cut with BamHI-isolated arms (Maniatis et al.,supra).

The resultant ligated DNA was then used to infect an E. coli DP50 strain(commercially available) on plates. Ten-thousand plaques were liftedonto non-sterilized nitrocellulose filters (Schleicher and Schuell,BA85, 132 nm diameter). The filters were denatured by contact with asolution of 0.5M NaOH, 1M NaCl. Then they were renatured in a solutionof Tris, pH 7.5, 3M NaCl and washed in 2×SSC. After renaturation, thefilters were baked for one hour at 80° C. in a vacuum oven. The filterswere then treated at 42° C. for five minutes in a prehybridizationbuffer consisting of 5×SSPE (20×SSPE is prepared by dissolving 174 g ofNaCl, 27.6 g of NaH₂ PO₄.H₂ O, and 7.4 g of EDTA in 800 ml of water withpH adjusted to 7.4 and volume adjusted to 1 liter; alternatively, SSPEis 0.18M NaCl, 10 mM NaPO₄, 1 mM NaEDTA, pH 7), 5×Denhardt's solution(1×Denhardt's solution--0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1%BSA), 0.1 mM ATP, 0.2 μg/ml sonicated salmon sperm DNA, 20% formamide,and 0.1% SDS. Then Probe 1, labeled at its 5' end with radioactivephosphorus as described below, was added to the prehybridization mixtureat a concentration of 2×10⁶ cpm per ml.

Probe 1 was 5' end labeled with 32P in a solution containing 0.05MTris-HCl pH 7.6, 0.01M MgCl₂, 0.005M dithiothreitol, 0.1 mM spermidine,0.1 mM EDTA, 35 μg/ml synthetic Probe 1, 4 mCi/ml gamma 32P ATP (about5000 Ci/mmol), and 10 U polynucleotide kinase for 30 min. at 37° C. Thelabeled DNA was phenol/CHCl₃ extracted, ethanol precipitated, andresuspended in water. Incorporated 32P was determined by spotting analiquot of the probe solution on a DEAE filter disc and washingextensively with 0.5N ammonium formate, followed by liquid scintillationcounting.

The filters were incubated with probe 1 for one hour at 42° C. Then thefilters were removed from the probe solution and washed three times in2×SSC plus 0.1% SDS at 37° C. for 15 min. The filters were exposed tox-ray film for three hours at -70° C. Positive plaques were chosen andgrown in liquid media for DNA.

After growth of the phage DNA, the phage were cleaved with severalrestriction enzymes. The resulting fragments were placed onnitrocellulose filters and treated with prehybridization buffer andProbe I as described above. The probe was found to hybridize to a 1.2 kbSalI to BamHI fragment on phage λ7. This fragment was isolated from λ7by cleaving with SalI and BamHI.

M13mp18 and M13mp19 bacteriophages, available from New England Biolabs,were cleaved with SalI and BamHI and the large fragment was isolated.The fragment from λ7 was ligated with the large fragments from M13mp18and M13mp19 such that the fragment from λ7 was under the control of thelac promoter. The resulting constructs were plated on JM101 on X-Galplates (Messing and Viera, Gene, 19: 269-276 [1982]). Colorless plaqueswere picked and single-stranded DNA templates containing the fragment inthe M13mp18 bacteriophage were sequenced by the dideoxy-chaintermination method of Sanger et al, supra, from the SalI site to theBamHI site and vice-versa, using a synthetic phage-specific primer.

After the determination of about 500 bp in each direction, new primerswere designed based on the determined sequence and the sequence wasextended an additional 500-bp. A 2-kb SalI-EcoRI fragment, shown byrestriction mapping to be adjacent to the SalI-BamHI fragment, wascloned into M13mp18 and the DNA sequence was determined for about 500bp. Samples were separated by electrophoresis on 5% polyacrylamide/8Murea "thin" gels. Gels were dried onto Whatman 3 MM paper and exposed toKodak x-ray film for varying lengths of time.

FIG. 3 shows the nucleotide sequence determined for the fragment fromthe SalI end to within about 300 bases of the BamHI end, as well as theimputed amino acid sequence of the 26-kd yeast ubiquitin hydrolase YUH-1and the positions of various restriction sites and the position ofprobe 1. FIG. 4 shows the direction of the sequencing.

FIG. 5 shows not only the predicted amino acid sequence of YUH-1, butalso its flanking region from HindIII to BamHI, based on the partialsequence of the 2-kb EcoRI-SalI fragment flanking the SalI-BamHIfragment containing lambda and yeast DNA. The location of the threesequenced peptides is indicated with starts. The 53-mer probe sequenceis shown below the correct DNA sequence. Mismatches are indicated aslower case letters. The probe is 87% identical with the correct DNAsequence.

The M13mp19 bacteriophage containing the SalI-BamHI hydrolase-encodinginsert was used to transform E. coli strain SR101 (commerciallyavailable), which was grown using as the culture media LB Broth. Theproduction of protein was induced by adding 1 mM of IPTG to the culturemedium.

Lysis of the SR101 cells was performed using the lysate proceduredescribed in the protein assay section herein for preparing the labeledprotein substrate for ubiquitin hydrolase from K5772.

The cell lysate was clarified by centrifugation and then assayed foractivity by the fusion polypeptide assay described above. The assayshowed detectable ubiquitin hydrolase activity by both assays,indicating that the DNA sequences cloned encoded hydrolase protein andwere induced by IPTG.

EXAMPLE II

DNA fragments encoding yeast ubiquitin were synthesized chemically on aDNA synthesizer using methoxyphosphoramidites. The yeast ubiquitin genewas synthesized connected to the gene coding for human (H2) relaxin Bchain of 32 amino acids, to yield the sequence shown in FIG. 1a, tohuman (H2) relaxin A chain of 24 amino acids, to prorelaxin, and to thehuman (H2) relaxin B chain of 33 amino acids to yield the sequence shownin FIG. 1b. Each fragment was designed so as to have a sticky-end XbaIand BamHI site at the ends. The prorelaxin construct is described inEuropean Pat. Pub. 260,149 published Mar. 16, 1988. The DNA sequence forA-chain relaxin is provided in EP Pub. No. 112,149, supra. Thedisclosures of both of these patent publications are incorporated byreference herein.

The synthetic genes were separately cloned into the large fragment afterXbaI and BamHI digestion of the plasmid trp 207-1*tetxap, described indetail in European Pat. Pub. 260,149. The ubiquitin-fusion polypeptidegene was ligated such that the ubiquitin fusion polypeptide gene wasunder the control of the trp promoter of the trp 207-1*tetxap plasmid.E. coli strain MM294 (commercially available) was transformed with theresulting plasmids and the synthesis of the fusion proteins was inducedby adding indoleacrylic acid to the culture medium. SDS-PAGE analysisusing silver staining under reducing conditions revealed a prominentprotein band. Upon Western blotting analysis this band reacted with anantibody directed against the appropriate relaxin moiety. The band wasfound to correspond to the correct molecular weight in each instance.

The culture of each transformant is then fermented, harvested, and runthrough an affinity chromatography column on which are immobilizedanti-ubiquitin monoclonal antibodies. The material bound to the columnis eluted and run through a column having the ubiquitin hydrolasepurified as described above adsorbed thereto. The eluent from the columnis passed through the affinity chromatography column on which areimmobilized the anti-ubiquitin monoclonal antibodies. The firstfractions containing separately the various relaxin polypeptides areobtained and pooled, free from the cleaved ubiquitin, the ubiquitinfusion polypeptide, and all cellular debris.

EXAMPLE III

The plasmid of Example II where the ubiquitin-relaxin A-chain syntheticDNA was inserted into the large fragment of XbaI and BamHI-digested trp207-1*tetxap is designated pTRP-Ubia. pTRP-Ubia was digested with XbaIand BamHI and the large fragment was isolated. The 1.2 kb SalI-BamHIfragment from the Charon 30 clone (Example I) was isolated and cleavedat the unique BanI site. Synthetic DNA encoding an E. coli ribosomebinding site sequence and a XbaI site at its 5' end (its sequence shownin FIG. 6) was ligated to the BanI site so that the 5' untranslated endof the cloned ubiquitin hydrolase gene was replaced by the binding site.The resulting XbaI-BamHI fragment was cloned into the large fragement ofpTRP-UbiA such that the gene was placed under the control of the trppromoter of the plasmid. The construction of this plasmid, designatedpTRP-YUH, is shown in FIG. 6.

pTRP-YUH was transformed into E. coli strain MM294 and grown in eitherLB media or M9 minimal casamino acids media (lacking tryptophan) tosaturation at 37° C. Samples were lysed at 90° C. in SDS sample bufferas described herein and electrophoresed through a 10% SDS polyacrylamidegel, followed by staining in Coomassie brilliant blue. FIG. 7 shows theresults. Lane (a) is growth of the E. coli strain in M9 minimal casaminoacids media (lacking tryptophan) and lane (b) is growth in LB media.

The E. coli transformed with pTRP-YUH in M9 medium overproduced the26-kd ubiquitin hydrolase, as determined from the prominent band on theSDS-PAGE gel shown in FIG. 7. The same plasmid in LB media did notproduce a 26-kd ubiquitin hydrolase band.

The ubiquitin hydrolase was isolated and purified as follows: About 60 gof E. coli cells was resuspended in 120 ml of Buffer A. The cellsuspension was sonicated in a sonicator for 3.5 minutes and clarified bycentrifugation. The clarified supernatant was loaded directly onto aDEAE Sephacel column (2.5×16 cm) equilibrated with Buffer A containing0.1M NaCl. The column was washed with Buffer A containing 0.1M NaCl,then developed with a linear gradient of 0.1M NaCl to 0.5M NaCl inBuffer A and then washed with Buffer A containing 0.5M NaCl. Theactivity was located by the cysteine release assay and pooled (60 ml).Enough ammonium sulfate was added to the pooled enzyme solution to givea 10% (w/v) final concentration.

The enzyme solution was then loaded onto a phenyl-Sepharose column(2.5×12 cm) equilibrated with Buffer A and 10% (w/v) ammonium sulfate.After a wash with Buffer A and 10% (w/v) ammonium sulfate, the columnwas developed with a decreasing gradient of 10% to 0% (w/v) ammoniumsulfate in Buffer A. The hydrolase activity co-eluted with the majorprotein peak and was essentially pure after this step, as determinedfrom SDS-PAGE analysis, shown as lane 3 in FIG. 7. All active fractionswere pooled and dialyzed versus Buffer A and 50% glycerol for storage.

The purified protein was capable of cleaving ubiquitin protein fusionsand was active in the cysteine release assay, using the assays describedin Example I.

EXAMPLE IV

This example illustrates the ability of the ubiquitin hydrolase tocleave proteins in vivo in E. coli.

A DNA fragment containing the trp promoter and YUH-1 gene from plasmidpTRP-YUH shown in FIG. 6 was cloned into lambda gt11 phage. Theconstruction of lambda gtll TRP-YUH is shown in FIG. 8. The recombinantphage was used to lysogenize T7 RNA polymerase-containing strain K5772.The resulting strain, designated E. coli strain K5808, which contains agene coding for ubiquitin hydrolase in its genome, was deposited in theAmerican Type Culture Collection under ATCC No. 53,832 on Nov. 30, 1988.

A plasmid pT7-3UbiAP containing a ubiquitin-relaxin A fusion protein wasprepared as depicted in FIGS. 9-13, starting with pT7-12 and ptrpST2HGH.

Construction of pT7-12ST2HGH (FIG. 9)

Plasmid pT7-12 (described in Example I) was digested with HincII andBamHI and the large vector fragment isolated. Plasmid ptrpST2HGH (EP177,343) was digested with HpaI and BamHI and the small hGH codingfragment isolated. The two fragments were ligated to producepT7-12ST2HGH.

Construction of pT7-12ST2TPA-1 (FIG. 10)

Plasmid pT7-12ST2HGH was digested with XbaI and BamHI and the largevector fragment isolated. Plasmid pΔRIPA.sup.• (EP 93,619, containingthe E. coli trp promoter/operator and the human t-PA gene) was digestedwith BstXI and BamHI and the small fragment (the t-PA gene) isolated.The same plasmid was also cleaved with BstXI and PstI and the 383-bpfragment isolated. These three fragments were ligated with twodouble-stranded synthetic DNA fragments A (encoding the N-terminus ofthe STII signal) and B (encoding the C-terminus of the STII signal fusedto the N-terminal seryl residue of t-PA) to yield pT7-12ST2TPA-1.##STR5##

Construction of pAPST2IFN-γΔNdeI-AvaI (FIG. 11)

Plasmid pIFN-γ (tetra-Ser) (de la Maza et al., Infection and Immunity55:2727 [1987]) was digested with AvaI and then partially digested withNdeI. The vector was treated with the Klenow fragment of DNA polymeraseI to repair the sticky ends and then isolated and ligated to itself toprovide pTetraSer ΔAN. Plasmid pAPH-1 (P. Gray et al., Gene 39:247[1985]) was digested with EcoRI and XbaI, the 400 basepair EcoRI-XbaIfragment was isolated and ligated into EcoRI- and XbaI-digestedpTetraSer ΔAN to provide plasmid pAP tetraSer. Plasmid pAP tetraSer wasdigested with XbaI and NdeI and the vector was isolated. Syntheticoligonucleotide fragments of sequence shown in FIG. 11 were kinased,annealed and ligated into the XbaI-NdeI vector obtained from plasmid pAPtetraSer to provide plasmid pAPST2IFN-γΔNdeI-AvaI, which contains apromoter from the alkaline phosphatase gene (Y. Kikuchi et al., NucleicAcids Res. 9:5671 [1981]) and the E. coli secretory signal of heatsignal enterotoxin II (R. N. Picken et al., Infection and Immunity42:269 [1983]).

Construction of pT7-3ST2TPA (FIG. 12)

pT7-12ST2TPA-1 is digested with XbaI and BamHI and the small fragmentisolated. The same plasmid is digested with XbaI and PvuII and a 96-bpfragment isolated.

pAPST2IFN-γΔNdeI-AvaI is cleaved with HindIII, treated with Klenow DNApolymerase I to fill in the sticky end, and cleaved with BamHI, and thelarge fragment is isolated.

The XbaI-BamHI small fragment, the 96-bp fragment, and the largefragment from pAPST2IFN-γΔNdeI-AvaI are ligated together to give theplasmid pT7-3ST2TPA.

Construction of pT7-3UbiAP (FIG. 13)

pT7-3ST2TPA is digested with XbaI and BamHI and the large fragmentisolated. pTRP-UbiA was digested with XbaI and BamHI and the smallfragment was isolated. This small fragment was ligated to the largefragment from pT7-3ST2TPA. The resulting plasmid, pT7-3UbiA, wasdigested with DraIII and PvuII and ligated to the synthetic DNA fragmentindicated in FIG. 13 to yield pT7-3UbiAP.

Construction of pT7-12UbiB

pT7-12ST2TPA-1 is digested with XbaI and BamHI and the large fragment isisolated and ligated to a synthetic DNA fragment encoding theubiquitin-relaxin B-29 fusion protein and having XbaI and BamHI stickyends, to produce pT7-12UbiB.

Transformation and Cleavage

Strain K5808 was transformed with either of pT7-3UbiAP or pT7-3UbiB. Inaddition, strain K5772 was transformed with a plasmid encoding relaxinA-chain alone (under the control of pT7 promoter) as a control, and thecell supernatant was analyzed by SDS-PAGE gel. Also the supernatant ofthe relaxin A-chain fusion treated with ubiquitin hydrolase purifiedfrom E. coli from Example III was also analyzed.

The K5772 and K5808 strains were grown in M9 minimal salts mediacontaining 50 μg/ml of each common amino acid except methionine,cysteine, and tryptophan, and in 50 μg/ml of ampicillin to maintain theappropriate plasmid. Incubations were at 37° C. for K5772 derivativesand 32° C. for K5808 derivatives. Cultures were grown to an OD of 0.4,at which time IPTG was added to a concentration of 1 mM and incubationwas continued for 30 min. Rifampicin was added to 200 μg/ml and theculture was incubated an additional 30 min. 35S-cysteine was added (10μCi/ml, 600 Ci/mmol), and the culture was pulse-labeled for theindicated time. Samples were either immediately lysed in SDS samplebuffer (10% glycerol, 5% beta-mercaptoethanol, 2.3% SDS, 0.0625 Tris-HClpH 6.8, 0.04% Bromphenol Blue) at 90° C. for 5 min., then placed on ice,or the labeled cells were rapidly pelleted in eppendorf tubes and frozenin ethanol/dry ice. When pulse-chase experiments were performed,chloramphenicol (100 μg/ml) was added immediately after the pulse andincubation was continued, with pulse-chase samples taken at 10, 20 and30 minutes. Thus, the polypeptides, labeled in vivo by the T7 RNApolymerase, were followed by pulse chase and analyzed on a reducingSDS-PAGE gel.

The SDS-PAGE results are shown in FIG. 14, where Lane (a) is the cellsupernatant of labeled relaxin A fusion in E. coli strain K5772, (b) isthe supernatant of labeled relaxin A fusion treated in vitro withpurified ubiquitin hydrolase from E. coli, (c)-(f) is relaxin A fusionlabeled in strain K5808 containing the ubiquitin hydrolase gene, with(d)-(f) chased for 10, 20, and 30 min., respectively, (g)-(j) is relaxinB fusion labeled in strain K5808, with (h)-(j) chased for 10, 20, and 30min., respectively. The positions of ubiquitin monomer and the relaxin Apolypeptide are indicated.

The strain K5772 lacking the YUH-1 gene as well as the integrated lambdaphage produced only the fusion proteins. However, the K5808 straincontaining the YUH-1 gene produced fusion proteins that are chased intoubiquitin-sized polypeptide. The carboxylterminal extension polypeptiderelaxin A-chain was apparently degraded, as was the relaxin B-chain.

EXAMPLE V

This example shows the effect of changing the amino acid at position 76of ubiquitin or at the N-terminus of the polypeptide fused to theubiquitin (position 77).

The ubiquitin gene has a convenient unique SalI site 5' to the uniqueDraIII site that was used to link DNA encoding fusion proteins. The DNAfragments given below were linked to the remainder of the nucleotidesneeded to reconstitute the ubiquitin 3' end and have a SacII siteinserted. To this end, pT7-12UbiAP containing DNA encoding ubiquitinfused to relaxin A was cleaved with SalI and BamHI and ligated with oneof the following synthetic DNA fragments having a SalI sticky end at the5' end and a BamHI sticky end at the 3' end on the top strand.(Nucleotides contributed by the vector that encode the remainder of thepolypeptide are underlined.) pT7-12UbiAP is prepared by cleavingpT7-12ST2TPA-1 with XbaI and BamHI, isolating the large fragment,cleaving pT7-3UbiAP with XbaI and BamHI and isolating the smallfragment, and ligating these two fragments together. ##STR6##

Another set of constructions is prepared by cleaving pT7-3UbiAP withDraIII and BamHI, isolating the large fragment, and ligating it to asynthetic DNA fragment encoding one of the following polypeptides:

(Asp 77) ubiquitin-Asp-amino acids 2-29 of relaxin B-chain.

(Gln 77) ubiquitin-Gln-amino acids 2-24 of relaxin A-chain.

Also (Glu 77) [ubiquitin-Glu-pentapeptide-amino acids 2-33 of relaxinB-chain] was prepared as described above (FIG. 1b and Example I).

In addition, plasmids were constructed such that the synthetic DNA forthe fusion polypeptide, driven by the pT7 promoter, encoded a methionineor cysteine residue at position 77 (at the N-terminus of the fusedpolypeptide).

Strain K5772 was transformed with each of the above plasmids, theprotein substrates were labeled, and the fusion protein in vitrocleavage assay was performed as described in Example I.

Efficient cleavage was achieved for the Glu 77, Asp 77, Gln 77, Cys 77,Gly 77, and Met 77 constructions. If proline was inserted at thisposition 77, however, no cleavage was observed, consistent with the invivo results of Bachmair et al., Science, 234: 179-186 (1986). Inaddition, substitution of valine or cysteine at position 76 of ubiquitin(the C terminus) blocked cleavage, showing the importance of the glycineresidue at position 76.

EXAMPLE VI

Finley et al., Cell, 48: 1035-1046 (1987) found that deletion of theyeast penta-ubiquitin gene rendered the cell sensitive to environmentalstress. Removal of the YUH-1 gene should therefore produce the same or amore severe phenotype, because the cell should have no way to generateubiquitin monomer, and therefore be ubiquitin minus.

Therefore, the YUH-1 gene was interrupted with the Ura3 gene asdescribed below. The final plasmid, pYUH::URA3, was prepared frompCGY379, the construction of which is described below, and pT7-3ST2TPAdescribed above.

pCGY379 was prepared by cleaving YEp24 (New England Biolabs) withHindIII and isolating the 1166-bp fragment. The fragment was ligatedwith the synthetic linkers described in FIG. 15. pUC119 (commerciallyavailable) was cleaved with SalI and ligated with the 1166-bp fragmenthaving the linkers. The resulting plasmid was pCGY379, and itsconstruction is illustrated in FIG. 15.

pYUH::URA3 was prepared as follows: pTRP-YUH (described above) wascleaved with SalI and EcoRI and the small fragment containing the YUH-1gene was isolated. pT7-3ST2TPA was cleaved with EcoRI and SalI and thelarge vector fragment was isolated. These two fragments were ligated toobtain plasmid pYUH1. pYUH1 was cleaved with EcoRV. pCGY379 was cleavedwith SmaI and the small fragment (1.1 kb) containing the yeast URA3 genewas isolated. This SmaI fragment and the cleaved pYUH1 plasmid wereligated, and the ligated product was cleaved with EcoRV, to yieldpYUH::URA3 containing the URA3 gene within the YUH-1 gene. Theconstruction is shown in FIG. 16.

The pYUH::URA3 plasmid was cleaved with SalI and EcoRI. The smallfragment, a 1.95-kb fragment, is shown in FIG. 17, along with theSalI-EcoRI 847-bp fragment containing the uninterrupted YUH-1 gene usedas a hybridization probe.

A diploid ura3/ura3 yeast auxtroph (a/α ura3-52) may be prepared bymating the haploids DBY747 (MAT a ura3-52) and DBY746 (MAT α ura3-52) bya standard mating technique. These haploids are available from the YeastGenetic Stock Center in Berkeley, Calif. The resulting diploid may betransformed with the 1.95-kb fragment as described by Rothstein, Meth.Enzymol., 101: 202-211 (1983). URA⁺ diploids are selected andsporulated. Rothstein, supra. The spores are dissected and tested forgrowth and URA phenotype. All spores, whether URA⁺ or URA⁻, are viableand grow into haploid yeast colonies. DNA is prepared from the parentaldiploid strain, the URA⁺ diploid, a URA⁺ haploid spore from the URA⁺diploid, and a urahaploid spore from the URA⁺ diploid, cleaved with SalIand EcoRI, and Southern blotted using the 847-bp SalI-EcoRI fragmentfrom the YUH-1 gene as a probe, labeled as described below, to screenfor the insertion of the URA3 gene into the YUH-1 gene.

The 847-bp SalI-EcoRI DNA fragment is labeled by random primed DNAsynthesis. A mixture containing 8 μg/ml DNA in 10 mM Tris-HCl pH 7.4, 10mM MgCl₂, 100 μM dATP, dGTP, and TTP, 4 mCi/ml alpha 32P dCTP, and 40μl/ml calf thymus primer is heated to 100° C. for 2 min., then cooled onice. Klenow enzyme (400 U/ml) is added and incubation is for one hour atroom temperature. The mixture is phenol/CHCl₃ extracted and ethanolprecipitated. Incorporated radioactivity is determined as described inExample I. The labeled probe is heated to 100° C. for 5 minutes andchilled on ice prior to addition to hybridization reactions. Thehybridization of the probe is carried out as described in Example I.

The resulting Southern blots are shown in FIG. 18, where Lane (a) is theparental strain (ura⁻ diploid), (b) is the URA⁺ diploid, (c) is a URA⁺haploid, and (d) is a ura⁻ haploid.

The 847-bp parental SalI-EcoRI fragment is seen in the parental diploid,URA⁺ diploid, and ura⁺ haploid. The 2-kb insertion fragment is onlypresent in the URA⁺ haploid and the URA⁺ diploid. These experimentsconfirm the presence of URA3-interrupted YUH-1 gene in the haploidspores as well as in one chromosome of the diploid parentaltransformant.

Extracts of the YUH-1-negative URA⁺ diploid and haploid clones areassayed for ubiquitin-protein in vitro cleavage activity as described inExample I. The results show that the YUH-1-negative clones containnearly wild-type levels of enzyme. Since the URA3 gene interrupts theYUH-1 gene at the 35th amino acid and no AUG translational start codonis found in the YUH-1 gene until the very C-terminus, no active proteincould be made from the split YUH-1 gene. Therefore, there exists atleast a second gene, YUH-2, which codes for a second yeast ubiquitinhydrolase. This gene is not related to YUH-1 at the DNA sequence levelbecause no second band of hybridization is seen on the FIG. 18 Southernblot of DNA from the YUH-1-negative URA⁺ haploid when the YUH-1 gene isused as a probe at low stringency. The gene YUH-2, therefore, encodes asecond yeast ubiquitin hydrolase.

This second yeast ubiquitin hydrolase protein is cloned and expressed byfirst fermenting the URA⁺ haploid whose Southern Blot is shown in Lane(c) and purifying and partially sequencing the hydrolase containedtherein (for purposes of designing a probe) using the method describedin Example I, Sec. II. Once at least a partial sequence is obtained, itmay be used to prepare a synthetic probe, which is used to screen ayeast library as described in Example I, Sec. III. The DNA fragmenthybridizing to the probe is isolated from the relevant phage, clonedinto M13mp19 and/or M13mp18 bacteriophage, and sequenced as described inExample I. The hydrolase-encoding fragment is then linked to a suitablepromoter such as trp, and to a ribosome binding site, transformed intoan E. coli strain and induced for expression as described, for example,in Example III for YUH-1.

SUMMARY

In summary, a gene (YUH-1) has been cloned from the yeast, Saccharomycescerevisiae, that codes for a catalytic activity that processesubiquitin-protein fusions. The gene codes for a 26-kd protein and, asfor most yeast genes, contains no introns. The YUH-1 gene can beoverexpressed in E. coli in active form and purified to homogeneity andthe enzyme is capable of cleaving ubiquitin fusions intracellularly inE. coli.

When isolated from yeast, ubiquitin hydrolase purifies as a complexcontaining at least two other proteins.

Gene interruptions of the YUH-1 gene are not lethal to haploid yeast.Indeed, the levels of ubiquitin hydrolase as assayed by cysteine releaseassay are near normal in such strains. The yeast cell appears to containa second ubiquitin hydrolase gene YUH-2.

The yeast ubiquitin hydrolase is specific for cleaving the peptide bondfollowing gly76 of ubiquitin fusion proteins. Alteration of a singleamino acid in the ubiquitin moiety at position 76 appears to inhibitcleavage. However, the enzyme is relatively insensitive to the size ofthe C-terminal extension or to the residue following the cleavage site,except for proline.

In vivo synthesis of ubiquitin fusion proteins that are purified andprocessed in vitro will allow the synthesis of proteins with specifiedamino termini. This process may be of particular utility for makingsmall peptides that, due to their size, may be rapidly degradedintracellularly. Because ubiquitin hydrolase is also active in cleavingubiquitin from ubiquitin-protein conjugates, purified hydrolase isextremely useful for deubiquitinating eukaryotic proteins in vitro priorto performing activity assays and/or sequence determination, because thepresence of ubiquitin may interfere with these processes.

What is claimed is:
 1. A bacterial host cell having DNA encoding a yeastubiquitin hydrolase as shown in FIG. 5 integrated into theirchromosomes.
 2. The host cell of claim 1 that is E. coli.
 3. The hostcell of claim 2 having the characteristics of Strain K5808 cellsdeposited under ATCC No. 53,832.
 4. The host cell of claim 1 that istransformed with an expression vector comprising a nucleotide sequenceencoding a ubiquitin-polypeptide fusion wherein the polypeptide is fusedto the C-terminus of the ubiquitin and wherein the polypeptide containsany amino acid except proline at its N-terminus.
 5. The host cell ofclaim 4 wherein the polypeptide is heterologous to the host cell.
 6. Thehost cell of claim 5 wherein the polypeptide is a human polypeptide. 7.The host cell of claim 6 wherein the polypeptide is relaxin or insulinA-chain or B-chain, prorelaxin, proinsulin, an interferon, aninterleukin, a growth hormone, a nerve growth factor, a transforminggrowth factor, an insulin-like growth factor, or DNase.